Review Article Biodegradable Polyphosphazene Biomaterials...

Review ArticleBiodegradable Polyphosphazene Biomaterials forTissue Engineering and Delivery of Therapeutics

Amanda L Baillargeon1 and Kibret Mequanint12

1 Graduate Program of Biomedical Engineering Faculty of Engineering The University of Western OntarioLondon ON Canada N6A 5B9

2Department of Chemical amp Biochemical Engineering Faculty of Engineering The University of Western OntarioLondon ON Canada N6A 5B9

Correspondence should be addressed to Kibret Mequanint kmequaniuwoca

Received 1 February 2014 Accepted 29 March 2014 Published 29 April 2014

Academic Editor Deon Bezuidenhout

Copyright copy 2014 A L Baillargeon and K Mequanint This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Degradable biomaterials continue to play a major role in tissue engineering and regenerative medicine as well as for deliveringtherapeutic agents Although the chemistry of polyphosphazenes has been studied extensively a systematic review of theirapplications for a wide range of biomedical applications is lacking Polyphosphazenes are synthesized through a relatively well-known two-step reaction scheme which involves the substitution of the initial linear precursor with a wide range of nucleophilesThe ease of substitution has led to the development of a broad class of materials that have been studied for numerous biomedicalapplications including as scaffold materials for tissue engineering and regenerative medicine The objective of this review is todiscuss the suitability of poly(amino acid ester)phosphazene biomaterials in regard to their unique stimuli responsive propertiestunable degradation rates and mechanical properties as well as in vitro and in vivo biocompatibility The application of thesematerials in areas such as tissue engineering and drug delivery is discussed systematically Lastly the utility of polyphosphazenes isfurther extended as they are being employed in blend materials for new applications and as another method of tailoring materialproperties

1 Introduction

Over the past few decades tissue engineering and regenera-tivemedicine have become significant areas of research due totheir potential to fix or replace damaged tissues and prolonglife [1 2] Tissue engineering and regenerative medicineincorporate knowledge from the areas of biology materialsscience and engineering to repair restore and regenerateliving tissues that may have been compromised by diseaseinjury or other means [3 4] Combining the expertise fromthese disciplines along with the development and applica-tion of biomaterials cells and bioactive molecules such asgrowth factors tissue-engineered products and regenerativemedicine strategies that are capable of extending lifespansand overcoming numerous health problems is made possible[3 5 6] Not surprisingly the development of suitablebiomaterials including a variety of polymers and ceramics

which are critical for the success of tissue engineering andregenerative medicine is being explored [7 8] Dependingon the target tissue to be engineered the biomaterial thatis used must exhibit several key characteristics such as bio-compatibility biostability or biodegradability and suitablemechanical properties (eg tensile strength and compressionresistance)

Biomaterials for tissue engineering must be biocompati-ble since they eventually must be implanted into the patientand a prolonged immune response would be problematic [9]Natural polymers such as chitosan collagen and gelatin areknown to be highly biocompatible and therefore have beenextensively studied as biomaterials for tissue engineeringand other biomedical applications [4 10] Their main draw-backs are their inadequatemechanical strength uncontrolleddegradation rates and poorly defined structure [10 11] Thishas lead researchers to investigate synthetic polymers as

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 761373 16 pageshttpdxdoiorg1011552014761373

2 BioMed Research International

an alternative to natural materials Biodegradability is adesirable feature of a biomaterial used in tissue engineeringsince the goal is that it acts as a temporary scaffold holdingthe growing tissue in place until the natural extracellularmatrix has sufficiently developed Beyond that point the scaf-fold should breakdown into nontoxic degradation productscapable of being disposed of by the body leaving only thenewly formed tissue There are a wide variety of syntheticbiodegradable polymers that have been and continue to beexplored including polyesters polyanhydrides polyacetalsand poly(120572-amino acids) [10] Despite their improvementover natural polymers with regard to degradation andmechanical properties synthetic polymers have their ownlimitations A common problem of synthetic polymers suchas poly(lactic acid) (PLA) is the formation of acidic productsduring the degradation process which leads to diminishedmechanical strength of the material and compromised cellfunction in the acidic environment [12ndash14] The quest forbiomaterials with tunable degradation rates and mechanicalproperties which also maintain cell function and lack theformation of toxic degradation products is an active area ofresearch [15 16]

Sustained research towards new biomaterials for tissueengineering and regenerative medicine applications has ledto the utilization of polyphosphazenes as a class of novelmaterials Polyphosphazenes are comprised of an inorganicbackbone of repeating phosphorus and nitrogen atoms withalternating single and double bonds (Figure 1 Structure 1c)[6 17ndash19] Extending from each of the phosphorus atoms aretwo organic side chains which can range from alkoxy andaryloxy substituents to amino acids giving a large variety ofpotential polymers [5 18 20 21] Changing the organic sidegroups and their ratios if multiple different side groups areattached to the same polymer backbone allows substantialtunability of the physical and degradation properties ofthe material [18 22 23] Therefore altering the organicsubstituents can be quite useful in tailoring the mechanicalproperties and degradation rates of the biomaterial to suitthe desired tissue engineering application such as bone tissueor blood vessels which require drastically different physicalproperties [24 25]

2 Synthesis of Polyphosphazenes

The synthesis of polyphosphazenes such as those shown inFigure 1 is typically via a two-step reaction beginning withthe thermal ring opening polymerization of hexachloro-cyclotriphosphazene (1a) the cyclic trimer to the linearpoly(dichlorophosphazene) (1b) precursor Next the organicside chains are attached to the polymer backbone througha nucleophilic macromolecular substitution of the organicsubstituents for the phosphorus-bound chlorine atoms [1821 26] The following two sections succinctly will describethe individual steps of polyorganophosphazene synthesisshowing the vast range of materials that can be generated

21 Thermal Ring Opening Polymerization-Bulk PhaseAlthough the thermal ring polymerization of the trimer (1a)

CH3

CH3

N

N

N

N

N

P

P

P

P

P

O

O

O

OO

O

O

O

O

O

O

O

HO

HOHO

HO

H

H

HH

H

HH

H HH

R

R

R

R

OR998400

OR998400

OR998400

NH

NH

NH2

NH2

OH

OH

OH

OH

2

3

451c

R998400O

n

n

n

n

n

Figure 1 Structures of various polyphosphazenes includingsteroidal substituents (2) carbohydrates (3) amino acid esters(4) and side chain-bound amino acid esters (5) to name a fewAdapted from [5] by permission of the Royal Society of Chemistry(httpdxdoiorg101039B926402G)

to linear poly(dichlorophosphazene) was attempted inthe late 1800s by H N Stokes a useful material thatwas soluble and capable of being functionalized was notrealized until the 1960s The initial thermal ring openingpolymerization performed by Stokes lead to a product thatwas insoluble due to crosslinking and that was readilysusceptible to hydrolysis when exposed to moisture [18]In 1965 Allcock and Kugel [27] were able to synthesislinear poly(dichlorophosphazene) through a well-controlledthermal ring opening polymerization from the cyclic trimerhexachlorocyclotriphosphazene according to Scheme 1 Theproduct obtained was soluble allowing it to be modifiedfurther by macromolecular substitution of the reactivePndashCl bonds with organic and organometallic nucleophilesThe thermal ring opening polymerization techniquedeveloped by Allcock et al is the most commonly usedroute to prepare the linear poly(dichlorophosphazene)precursor [5 18] A typical process involves reactingpurified hexachlorocyclotriphosphazene trimer at 250∘Cover 5 days in an evacuated polymerization tube Atthis point soluble poly(dichlorophosphazene) has beenformed that can be purified and functionalized via

BioMed Research International 3

P

P P

P P P P

PN

N N

N N N

N

N

ClCl

Cl

Cl

Cl Cl

ClCl

250∘C 120h

NHR

NHR

NR2

NR2

HN

R 2

MR

OR

OR

1a 1b

H 2NR

R

R

NaOR

n

n n n n

Scheme 1 Scheme showing the synthesis and functionaliza-tion of poly(dichlorophosphazene) (1b) in the overall synthesisof polyphosphazenes from hexachlorocyclotriphosphazene (1a)Reproduced from [5] by permission of the Royal Society of Chem-istry (httpdxdoiorg101039B926402G)

the macromolecular substitution reaction (Scheme 11ararr 1b) [18] Despite the success of the bulk phasethermal ring opening polymerization in lab scale synthesesthis method is not economically feasible for large-scaleproduction of polyphosphazene materials Alternativemethods which will not be discussed in this reviewincluding solution phase thermal ring opening [28] livingcationic [29ndash32] and one-pot De Jaeger [33] polymerizationtechniques have been reported

22 Functionalization of the Poly(dichlorophosphazene) Pre-cursor Once high molecular weight linear poly(dichloro-phosphazene) is synthesized the polymer can be modifiedby substituting the phosphorus-bound chlorine atoms withorganic side groups The polymer undergoes a macromolec-ular substitution (Scheme 1 reaction of compound 1b intofour potential polyphosphazene structures) when subjectedto organic and organometallic nucleophiles forming a largeclass of polyphosphazenes as shown in Figure 1 [18 26]All of the polyphosphazenes shown in Figure 1 have onetype of side chain throughout the entire polymer althoughit has been shown that cosubstituted materials with well-defined ratios of side chains are possible by controlling theamount and order addition of the nucleophiles [18 21 34]The modification of type and ratios of the side chains ofthe polymer affords the ability to fine-tune degradation ratesand physical properties based on these substituents which isimportant to the synthesis of a biomaterial suitable for tissueengineering and therapeutic delivery [23]

3 Suitability of Polyphosphazene Biomaterials

In order for polyphosphazenes to be considered a suitablebiomaterial they must be compatible with the biologicalenvironment they are intended to interact with [5 35 36]They must also be either biostable or biodegradable intonontoxic degradation products Bioerodible biomaterials areusually preferred since they leave only the natural tissue oncethe material has degraded eliminating the long-term risk

of immune response and potential negative outcome [1023 35 37ndash47] Lastly the biomaterial must have mechanicalproperties that match or closely resemble those of the naturaltissue so that issues such as compliancemismatch a commonproblem for example in vascular tissue engineering arereduced [36 48] In the next few sections we summarizethe current understanding regarding the biocompatibilitybiodegradation and mechanical properties of polyphosp-hazene biomaterials The remainder of this review will focuson the suitability of polyphosphazenes mainly poly(aminoacid ester)phosphazenes as biomaterials due to their uniquetunability of degradation and mechanical properties makingthem useful in a wide range of biomedical applications as isshown in Figure 2 [5 21 49]

31 Stimuli Responsive Polyphosphazenes Wilfert et al [54]manipulated the biodegradability of polyphosphazenes andwere capable of developing materials with well-controlledpH responsive degradation rates that were also water sol-uble They synthesized materials with side chains includ-ing poly(ethylene oxide-copropylene oxide) (M-1000) alonevaline spaced M-1000 and glycine spaced M-1000 Degra-dation studies were performed by placing 20mg samples ofthe materials in deuterated water (D

2O) of varying pH (2 5

and 76) and monitoring changes in GPC traces 31P nuclearmagnetic resonance (NMR) spectra and ultraviolet-visible(UV-Vis) spectroscopy spectra It was shown that thematerialwithout any amino acid linkers degraded much more slowlythan those with valine or glycine linkers between the M-1000and the polyphosphazene backbone It was also demonstratedthat the polymers degraded more quickly in the presenceof acid with degradation rates of polyphosphazenes in pH2 being fully degraded in the time span of days whereaspolymers in neutral pH conditions (pH = 76) degradedmuch more slowly on the order of months and a substantialamount of the starting polymer remained after the 4 weekstudy period In order for these materials to be useful asdrug carriers with stimuli responsive degradation propertiesthey must also be biocompatible and it was shown thattheir degradation products did not significantly impact cellviability This study shows strong support of pH responsiveand water-soluble polyphosphazene-basedmaterials for theiruse in drug delivery applications

Thermoresponsive degradable polyphosphazenes con-taining lactic acid ester and methoxyethoxy ethoxy sidechains for use in biomedical applications were investigatedby Bi and coworkers [55] Three polymers were synthesizedwith different lengths of lactic acid ester alkyl chains rangingfrom ethyl to butyl The polymers with the butyl lacticacid ester had decreased lower critical solution temperatures(LCST) in comparison to those with ethyl esters indicatingthat they change from a solution to a precipitate gel atlower temperatures This is due to the fact that the butylchains are more hydrophobic than the ethyl esters causingthe materials to experience more hydrophobic interactions atlower temperatures Increased hydrophobic interactions leadto the exclusion of water from the polymer and the transitionof the polymer from a solution to a gelThe LCSTs of all three


(a) (b)

10 120583m 10 120583m

Tissue engineering scaffolds

100

80

60

40

20

0

0 5 10 15 20 25

Time (day)

Cum

ulat

ive a

mou

nt o

frel

ease

d sil

ibin

in (

) Drug delivery vehicles

PH 68 10mgmLPH 68 80mgmL


(c)

Polyphosphazene blends

PN

R

R

Stimuli responsive materials

0 sRoom temperature

Body temperature

10 s 30 s 60 s

0 s 10 s 30 s 60 s

n

Figure 2 Overview of several biomedical applications where polyphosphazenes have been shown to be useful biomaterials Reprinted from[50ndash53] with permission from Springer Science Business Media and Elsevier

materials were between 33∘C and 52∘C making them usefulin biological applications such as drug delivery Since thesematerials were being considered for in vivo applications theirdegradation characteristics and biocompatibility were alsotested MTT studies showed that the materials themselvesand their degradation products were nontoxic to HepG2 andK562VCR cells The biocompatibility and capability to tunethe thermoresponsive properties of these polyphosphazenematerials indicate their utility as materials for biomedicalapplications such as drug delivery especially if localizedinjection is critical to the treatment plan

32 In Vitro and In Vivo Compatibility of Polyphosp-hazenes The cytocompatibility of amino acid ester func-tionalized polyphosphazene biomaterials was first studied byLaurencin et al [43] who compared rat primary osteoblastadhesion to poly[(ethyl glycinato) phosphazene] (PNEG)

with well-known poly(lactic acid-co-glycolic acid) (PLAGA)and poly(anhydrides) Data from this study showed thatthe osteoblast cells adhered to the PNEG material to thesame extent as the control materials for a period of 8hours The degradation of PNEG did not influence cellproliferation as it promoted cell growth to the same extentas the PLAGA control material In a follow-up study [56]similar experiments on other ethyl glycinatomethyl phe-noxy cosubstituted polyphosphazenes using MC3T3-E1 cells(osteoblast precursor cell line from mice) were conductedThe results from this study also suggested that cells respondedfavourably to polyphosphazene materials especially thosewith a high ratio of ethyl glycinato substituents and that celladhesion and proliferation characteristics were not dimin-ished in comparison to tissue culture plate and PLAGAcontrols The polymers with 50 and greater of ethyl gly-cinato substituents demonstrated improved cell growth incomparison to the tissue culture plate and the polymer


with 25 ethyl glycinato substitution was only slightly lesseffective than the tissue culture plate although all of thesewere better than the PLAGA control which has been widelyaccepted as a biocompatible material Studies on cosub-stituted amino acid ester-based polyphosphazenes contain-ing an ethyl alanato substituent along with aryloxy sub-stituents such as poly[(ethyl alanato)

1(ethyl oxybenzoate)

1

phosphazene] (PNEAEOB) and poly[(ethyl alanato)1(propyl

oxybenzoate)1phosphazene] (PNEAPOB) demonstrated that

neither PNEAEOB nor PNEAPOB posed a threat to cellgrowth in comparison to the controls as both materialswere capable of promoting cell adhesion and proliferation[47] Collectively the results of the above studies from theLaurencin laboratory are promising since cell adhesion andproliferation are not affected in comparison to materialsthat have previously been extensively studied for their effecton cell viability One possible drawback with these studieshowever is the cell sources (rat and mouse) that may notappropriately represent what would occur with primaryhuman cells since cell interactions with thematerials may notbe identical across species A more suitable cell type to usewould be human osteoblasts to get a better indication of howthe cells might react to the biomaterial in vivo with humansubjects

Gumusderelioglu and Gur [57] performed a studythat investigated the cytotoxicity of poly[bis(ethyl-4-aminobutyro)phosphazene] by analyzing the activity levelof succinic dehydrogenase (SDH) through an MTT assaymethod SDH plays a critical role in cellular metabolism andis therefore a good indicator of cytotoxicity [58] For theseexperiments extracts collected from the incubation of thepolymeric films with growth medium were added to 3T3 andHepG2 cells For the negative control extracts were collectedfrom a polyethylene centrifuge tube that was incubated withthe growth medium but lacked a sample of polymeric film Itwas shown that poly[bis(ethyl-4-aminobutyro)phosphazene]extracts did not significantly decrease cell viability in Swiss3T3 and HepG2 cells in comparison to negative controlsThematerial maintained cell viability as demonstrated by SDHactivity level greater than 80 of that of the control for alltime points and for both cell types This study was successfulin showing the cytocompatibility of the material with respectto 3T3 and HepG2 cells which are commonly used cell linesto study fibroblast and hepatocyte biology respectively Thefact that the cells studied are cell lines rather than primarycells is concerning since cell lines are known to grow welleven when conditions may not be ideal As such they maynot properly represent how the natural tissues which arenot composed of cell lines but rather of primary cells wouldrespond to the material Also it should be noted that the 3T3cells come from a Swiss mouse source and therefore just aswith the research performed by Laurencin et al the resultsmay not be indicative of how human cells would react to thematerial The cytocompatibility of electrospun matrices ofcosubstituted poly(amino acid ester)phosphazenes towardsrat endothelial cells was investigated by Carampin andcoworkers [59] They studied poly[(ethyl phenylalanato)

14

(ethyl glycinato)06phosphazene] for both cell adhesion and

growth properties in comparison to a fibronectin coated

polystyrene tissue culture plate as the control They foundthat the polymer only slightly improved cell adhesion(7 increase) in comparison to the culture plates but thatthe polymer enhanced growth of the adhered cells byapproximately 17 These results reinforced the notion thatpolyphosphazenes could act as a biocompatible material foruse in biomedical applications such as tissue engineeringAgain these results must be considered with caution asthey did not use human cells for their research Howeverthey did use primary cells which are more sensitive to theirenvironment than cell lines and are an improvement overcell line-based studies

All of the aforementioned studies involved only in vitroanalyses of the cytocompatibility of the polyphosphazenesdespite the fact that their end goal is to be used as abiomaterial in vivo Towards this end in vivo studies ofalanine-modified polyphosphazenes on rat and rabbitmodelsfor bone tissue engineering materials have been reported[5 47] In the rat model [47] subcutaneously implanted sam-ples were monitored for biocompatibility through immuneresponse Inflammatory responses were categorized as min-imal mild or moderate based on the accumulation ofimmune response cells (eg neutrophilsPMNs and lympho-cytes) at the implantation site It was observed that at 2weeks after implantation [poly(ethyl alanato) phosphazene](PNEA) induced a moderate inflammatory response thatinitially decreased to minimal at 4 weeks but then slightlyincreased to mild at 12 weeks As for poly[(ethyl alanato)

1(p-

methyl phenoxy)1phosphazene] (PNEAmPh) the material

caused a moderate inflammatory response at 2 weeks whichgradually decreased to a minimal response after 12 weeksThe poly[(ethyl alanato)

1(p-phenyl phenoxy)

1phosphazene]

(PNEAPhPh) material elicited a mild initial response at2 weeks which slowly decreased to a minimal responseat 12 weeks Overall the inflammatory responses for thePNEAmPh and PNEAPhPh were minimal suggesting thatthe materials are suitable for bone tissue engineering ThePNEA material triggered a greater inflammatory responsethan the two cosubstituted polyphosphazenes although theresponse decreased over the time span of the study suggestingthat it is a good candidate too All three materials elicitedimmune responses that were acute and did not pose a long-term threat to the animals

The potential utility of polyphosphazenes is not limitedto bone tissue engineering Langone et al [60] conducted invivo biocompatibility of polyphosphazenes as tubular nerveguides in rat models Comparative studies of poly[(ethylalanato)

14(imidazolyl)

06phosphazene] (PNEAIL) nerve

guides with traditional biostable silicone guides suggested theabsence of inflammatory response to the polyphosphazenematerial after 30 and 60 days of implantation Upon removalof the implanted nerve guide it was noted that the stumpsof the rat sciatic nerve which had initially been transectedhad reattached with components comprised primarily ofnerve fibre bundles akin the natural nerve tissue This invivo study suggested that PNEAIL was a biocompatiblematerial especially for use in nerve regeneration strategiesand highlighted its potential utility in the future Likethe work of Laurencinrsquos group [47] the materials were


analyzed in small animal models which do not behaveidentically to humans and therefore can only be used as aguideline towards how the materials might respond in aclinical sense

33 Biodegradability of Polyphosphazenes Since it is desirableto use biodegradable biomaterials for tissue engineeringtherapeutic delivery many research groups have studied thedegradation properties of polyphosphazenes [22 37ndash40 4244ndash47] Polyphosphazenes are attractive because they havebeen shown to degrade into nontoxic byproducts that areeasily metabolized by the body In the case of an amino acidester phosphazenes these hydrolytic degradation productsinclude the amino acid the corresponding alcohol of theester ammonia and phosphates [37] Unlike the acidicproducts produced from the hydrolysis of other polymersthe ammonia and phosphates act as a buffering systemand prevent fluctuations in pH which could otherwise bedetrimental to the tissue [61] Although the exact mechanismof degradation is not known there are several pathways thathave been proposed (see Scheme 2) [37] Overall the first twosteps of the degradation result in the hydrolysis of the esterof the amino acid forming an alcohol and detachment of theamino acid from the polyphosphazene backbone forming theamino acid itself The backbone of the polyphosphazene isthen hydrolyzed to phosphates and ammonia The formationof phosphates during the degradation process was verifiedthrough the addition of silver nitrate or zirconyl chloridewhich forms a yellow silver phosphate or white zirconylphosphate precipitate respectively [37] The amino acidsand ammonia degradation products can be demonstratedby ninhydrin test which detects ammonia and primary andsecondary amines whereas as 1H NMR spectroscopy can beutilized for detecting alcohols

Another important factor with regard to biodegradabilityis the rate at which the material degrades since this can limitpotential applications It is important when designing a scaf-fold that the material degrades at a rate that is similar to therate of tissue growth or therapeutic release rate depending onthe application For tissue engineering if the scaffoldmaterialdegrades too quickly there will be insufficient support for theunderdeveloped tissue and mechanical weakness will ensueIf the material degrades too slowly or not at all it may needto be surgically removed which could in turn damage theneotissue and cause problems with mismatched mechanicalproperties relative to the natural tissue [62] For therapeuticdelivery it is desirable to reduce burst release correspondingto rapid degradation and poor release corresponding to veryslow degradation In order to determine the degradationrates of poly(amino acid ester)phosphazenes the influence ofchanging the types and ratios of side chain substituents on thedegradation properties of the polymers is an important factorTable 1 provides an overview of the degradation studies thathave been performed on polyphosphazenes substituted withamino acid esters and other cosubstituents

In view of this the degradation rates of poly(amino acidester)phosphazenes with different amino acids and different

esters of the amino acids were studied in solution- and solid-state degradation although solid-state degradation is morerepresentative of how degradation would occur with in vivoscaffold materials and is the method that will be discussed[37] The effect of changing the ester group was investigatedusing glycine-based poly(amino acid ester)phosphazenesincluding poly[bis(methyl glycinat-N-yl)phosphazene](PNMG) poly[bis(ethyl glycinat-N-yl)phosphazene](PNEG) poly[bis(tert-butyl glycinat-N-yl)phosphazene](PNtBG) and poly[bis(benzyl glycinat-N-yl)phosphazene](PNBzG) In this systematic study the molecular weightdecline was in the order of PNBzG lt PNtBG lt PNEGlt PNMG with PNMG having the greatest decrease inmolecular weight This showed that as the hydrophobicityof the ester group increased (from methyl to benzyl) themolecular weight decline of the polymer decreased Thedecreased molecular weight decline is due to the inabilityof water to approach the polymer due to its hydrophobicityand therefore the hydrolysis of the material is limited Theeffect of changing the amino acid using poly[bis(methylglycinat-N-yl)phosphazene] (PNMG) poly[bis(methylalaninat-N-yl)phosphazene] (PNMA) poly[bis(methylvalinat-N-yl)phosphazene] (PNMV) and poly[bis(methylphenylalaninat-N-yl)phosphazene] (PNMF) showed that themolecular weight decline increased in the order of PNMF ltPNMV lt PNMA lt PNMGThis trendwas observed since thehydrophobicity of the polymer increased as larger nonpolarside chain amino acids like phenylalanine were incorporatedinto the polyphosphazene This study was a good initialdemonstration of the biodegradability and hydrolysisproperties of different poly(amino acid ester)phosphazenesalthough a more suitable degradation medium would bephosphate buffer solution (PBS) at 37∘C which is morerepresentative of the body fluid pH temperature and ionconcentrations

The effect of the types of side groups on the degradationrates of L-alanine cosubstituted polyphosphazenesspecifically PNEA poly[(ethyl alanato)

1(ethyl

glycinato)1phosphazene] (PNEAEG) PNEAmPh and

PNEAPhPh were also reported in a separate study [23] Asmay be expected the ethyl glycinato substituted phosphazene(PNEAEG) had the fastest molecular weight decline whereasthe biphenyl substituted phosphazene (PNEAPhPh) had theslowest molecular weight decline The PNEAEG materialhydrolyzed so quickly that molecular weight could not beevaluated beyond week two of the degradation study It wasnoted that the pattern of molecular weight decline showeda quicker degradation rate for the smaller more hydrophilicsubstituent polymers as compared to those substitutedwith large bulky hydrophobic substituents Compared toimidazolyl side groups increasing the amount of ethylglycinato groups increased the degradation rate of thepolymer indicating that the incorporation of less stericallyhindered more hydrophilic groups causes the polymers todegrade more quickly [56] The results of the study weresuccessful in demonstrating the tunability of degradationproperties of cosubstituted polyphosphazenes which is akey requirement in the development of a biomaterial fortissue engineering applications Overall this study effectively


P

P

P

NH

NH

NH

HN

H

N

N

O

O

O

O

O

O

O

O

O

O

O

O

O

O

R

R

R

R

N N

NN

N

N

N

N

P

P

P

P

minus OHH2O

H2O

H2O

H2O

H2OH2O

H2O

H2N

Phosphazanes

PhosphazanesP

P

P

P

NH

R

R

R

OH

OH

OH

Phosphates and ammonia


minus

H2Nminus

H2Nminus

∙ ∙

∙ ∙

∙ ∙ ∙ ∙

∙ ∙

∙ ∙

∙ ∙

∙∙

∙∙

∙∙

∙∙

∙∙

∙∙

∙ ∙

Hydroxyphosphazene

1

2

3

R998400

R998400

R998400

R998400

R998400

R998400

Scheme 2 Proposed degradation pathways for the hydrolysis of poly(amino acid ester)phosphazenes Reprinted with permission from [37]Copyright 1994 American Chemical Society

showed the ability to tune degradation rates of poly(aminoacid ester)phosphazenes through careful selection of sidegroup substituents One thing to consider when selecting sidegroups for biodegradable polyphosphazenes that incorporateamino acids is the degradation by natural enzymes found

in vivo If the enzymes are capable of recognizing the aminoacid enzymatic and hydrolytic degradation together mayincrease the degradation rate of the polymer as compared tohydrolysis alone Also if the enzymes in the native tissue arecapable of recognizing the polyphosphazene-bound amino


Table 1 Summary of in vitro degradation studies of poly(amino acid ester)phosphazenes and their cosubstituted polyphosphazenesThe esterrefers to the chain attached to the carboxyl terminus of the amino acid The detailed degradation profiles can be found in the cited papers

Amino acid Ester Cosubstituents Study length Reference(s)

Glycine

Methyl 35 days [37]

Ethyl

35ndash120 days [22 37 38]Alanine ethyl ester (50) 7 weeks [23]

p-Methyl phenoxy (25ndash90) 7 weeks [56]Ethyl-2-(O-glycyl)lactate (0ndash25) and phenylalanine ethyl ester (70) 120 days [22]

t-Butyl 5 weeks [37]Benzyl 5 weeks [37]

Alanine

Methyl 5 weeks [37]

Ethyl7 weeks [23 38]

p-Methyl phenoxy (50) 7 weeks [23]p-Phenyl phenoxy (50) 7 weeks [23]

Benzyl 7 weeks [38]Valine Methyl 5 weeks [37]Phenylalanine Ethyl 5 weeks [37]

acids their ability to interact with them may be stericallyhindered if bulky substituents are cosubstituted on thepolymer causing further complications in approximatingdegradation rates of poly(amino acid ester)phosphazenesfrom in vitro studies In vivo degradation studies showedsubstantial decline in molecular weight for the PNEAand PNEAmPh implants after 12 weeks 80 and 98respectively [47] PNEAPhPh on the other hand did notexperience as great of a molecular weight decline as the othertwo implants and had a molecular weight decline of only63 after 12 weeks This is presumably due to the increasedhydrophobicity of the biphenyl substituent which limits theapproach of water to the polymer backbone and thereforeslows its hydrolysis This study demonstrated the in vivobiodegradability of poly(amino acid ester)phosphazenesas well as their biocompatibility We should caution thatthe implant in this cited study was designed for bone tissueengineering applications and as such it is implanted intoa region of the rat where bone tissue is the predominanttissue type Naturally occurring enzymes which have thepotential to significantly influence degradation rates if theyrecognize the materials have different abundance acrossdifferent types of tissues Therefore if the poly(aminoacid ester)phosphazenes investigated in this study are tobe applied to other tissue engineering applications theirdegradation rates in those tissues may vary dramaticallyfrom those presented here due to differences in enzymaticdegradation The relative abundance of water in a tissuealso determines rates of hydrolysis and the materials couldtherefore show significantly different hydrolytic degradationrates in different tissues

Other studies were conducted on the effects of changingratios of substituents on the degradation rates of the polymers[41] Mass loss measurements following PBS incubationfocused on cosubstituted polyphosphazenes of ethyl 2-(O-glycyl) lactate and ethyl glycinato Decreasing the ratio ofethyl glycinato ethyl 2-(O-glycyl) lactate for materials withvarying side chain ratios between 100 ethyl glycinato

0 ethyl 2-(O-glycyl) lactate and 75 ethyl glycinato 25ethyl 2-(O-glycyl) lactate increased the mass loss rate of thepolymer This is due to the increased hydrolytic sensitivity ofethyl 2-(O-glycyl) lactate in comparison to ethyl glycinatowhich encourages polymer degradation and therefore massloss Even though mass loss is not a direct indication ofmolecular weight decline [23] it is still a good indicator ofthe relative degradation rates of the polymers and as suchit can be approximated that polymers with a higher ratio ofethyl glycinato substitution degrade less quickly than thosewith increased levels of ethyl 2-(O-glycyl) lactate This studywas useful in demonstrating the effect that varying ratios ofsubstituents with different hydrolysis-sensitivities and solva-tion properties has on the degradation rates of the polymerswhich can be useful for tailoring degradation properties ofcosubstituted polyphosphazenes according to their specificbiomedical applications Furthermore the degradation prop-erties of depsipeptide-substituted polyphosphazenes havealso been studied [63ndash65] Depsipeptides are short chainamino acid sequences that contain at least one ester linkagein place of an amide bond in the backbone of the peptidechain Although research on these types of polymers willnot be discussed in detail in this review it is importantto mention their role in developing suitable biomaterialsfor biomedical applications The reason that these polymershave been included in this review is that they are a goodpreliminary model for poly(amino acid ester)phosphazenesthat have been functionalized with bioactive moleculeswhich are typically proteins and short peptide chains Thedepsipeptide-type bonding in these functionalized polymerscomes from the amide linkages throughout the biomoleculeand a potential ester linkage through the carboxylate func-tionality of an amino acid side chain (eg aspartic andglutamic acid) The incorporation of these biomolecules cansignificantly enhance cellular interactions and biomimeticproperties of the materials making them better candidates asbiomaterials Research on these polyphosphazene materialshas shown their biodegradability therefore suggesting their


potential as biomaterials for tissue engineering and otherbiomedical engineering applications [41]

Taken together these studies have been able to demon-strate not only that poly(amino acid ester)phosphazenes arebiodegradable but also that their degradation rates can betuned by changing a variety of factors such as type and ratioof side groups The fact that bioerodability studies have beenperformed in vitro in body fluid simulating solutions and invivo in rat models suggests that these polyphosphazenemate-rials are suitable for use in tissue engineering applicationssuch as scaffold biomaterials as well as for other biomedicalapplications that require the use of degradable materials

34 Mechanical Properties of Polyphosphazenes In orderto produce clinically viable tissue-engineered products themechanical properties of the constructs must match theproperties of the natural tissues If the mechanical prop-erties such as compressive strength and tensile strengthare not comparable to those of native tissues problemswith mismatch arise which often lead to failure of thetissue-engineered construct [66ndash69] There have been onlya limited number of studies carried out to investigate themechanical properties of poly(amino acid ester)phosphazenematerials One such study was conducted by Sethuramanet al [70] who investigated the mechanical properties ofalanine-based polyphosphazenes for their application as bonetissue engineering biomaterials (Figure 3) For these studiespolyphosphazenes were compared with the current standardfor bone tissue engineering applications PLAGA (85 lacticacid 15 glycolic acid) Cylindrical discs of each polymerwere initially subjected to a compressive force of 1500 poundsper square inch (psi) for 15min and analyzed using a uni-axial compressive testing instrument set with the followingparameters 500N load cell and 10mmmin compression rateuntil material failure The compressive strengths of PNEAand PNEAmPh were comparable to that of PLAGA (349 plusmn57MPa) with compressive strengths of 4661 plusmn 1756MPaand 2498 plusmn 1126MPa respectively PNEAPhPh on theother hand had a compressive strength that was significantlyhigher than that of PLAGA due to the large aromatic groupsincreasing steric bulk and decreasing torsion of the polymerbackbone Together these increase the rigidity of thematerialand modulate its compressive properties Therefore it can benoted that the mechanical properties like the degradationproperties of polyphosphazene materials can be tailoredbased on their proposed applications by changing the sidegroup substituents The tensile strength and elasticity ofseveral L-alanine-based polyphosphazene materials (namelyPNEAEG PNEA PNEAmPh and PNEAPhPh) were deter-mined using microtensile testing techniques [70] It wasshown that increasing the steric bulk of the cosubstituentincreased both the tensile strength and elasticity of thematerial with more of an impact being observed as the sidechain is changed from a small amino acid such as glycine oralanine ethyl ester such as in PNEAEG and PNEA to largearomatic substituents such as in PNEAmPh and PNEAPhPhThis is because introducing large aryloxy substituents affectsthe glass transition temperature and molecular weight of the

Com

pres

sive s

treng

th (M

Pa)

PNEA PNEA50 mPh50

lowast

PNEA80PhPh20 PNEA50PhPh50 PLAGA 8515

100

80

60

40

20

0

Figure 3 Compressive strengths of alanine-based amino acid esterphosphazenes in comparison to poly(lactic acid-co-glycolic acid)[PLAGA (85 15)] The alanine-based polyphosphazenes presentedare poly[bis(ethyl alanato)phosphazene] (PNEA) poly[(50 ethylalanato) (50 methyl phenoxy)phosphazene] (PNEA

50

mPh50

)poly[(80 ethyl alanato) (20 phenyl phenoxy)phosphazene](PNEA

80

PhPh20

) and poly[(50 ethyl alanato) (50 phenyl phe-noxy)phosphazene] (PNEA

50

PhPh50

) The lowast indicates results thatare significantly different (119875 lt 005 119899 = 6) Reprinted from [70]with permission from Elsevier

polymer which in turn affects the mechanical properties ofthe material Overall this study shows how the mechanicalproperties of a polyphosphazenematerial can be tailored sim-ply through cosubstitution of large aromatic groups alongsideamino acid esters

Both of the above studies showed that changing the typesand ratios of side group chemistries of polyphosphazenematerials can modulate mechanical properties such as com-pressive strength tensile strength and elasticity Thereforethe mechanical properties of these materials can be tunedto suit the intended application making polyphosphazenesuseful as biomaterials in a wide range of different biomedicalapplications One concern here is that changing the sidegroups affects not only mechanical properties but also degra-dation rates and therefore adapting the side chains to obtainsuitable mechanical properties may cause the degradationrate of the material to be either too fast or too slow for theapplication which is undesirable As such it is suggested thatresearch into othermethods to controlmechanical propertiesthat do not influence erosion properties be developed Forexample it may be useful to investigate the effects of differentprocessing methods or scaffold preparation techniques (egelectrospinning versus solvent casting and particulate leach-ing) on mechanical properties

4 Tissue Engineering and Drug DeliveryApplications of Polyphosphazenes

Although many biomaterials have been previously investi-gated for tissue engineering applications there have been lim-itations to each such as acidic degradation products as wasdiscussed earlierTherefore once polyphosphazene materialswere studied and proven suitable for biomedical applicationsaccording to their biocompatibility biodegradability andmechanical properties they were implemented into tissue


engineering research The biomaterials are typically used toconstruct three-dimensional (3D) porous biodegradablescaffolds which temporarily support and direct tissue growthuntil the natural extracellularmatrix (ECM) develops A vari-ety of polyphosphazenes have been investigated as scaffoldmaterials for use in bone and skeletal tissue engineering[56 71ndash74] nerve guides [60] and blood contact materials[75] (eg coatings for implants and blood dialysis devices)

41 Bone Tissue Engineering The majority of research todate focused on polyphosphazenes as materials for boneand skeletal tissue engineering applications Laurencin andcoworkers have extensively studied these materials and theirinteractions with osteoblast type cells to determine theirsuitability for bone grafts and implants For example in 1996[73] they developed 3D and 2D matrices of amino acid-based polyphosphazene on which they seeded osteoblastcells They observed that the pores of the 3D constructsresembled in shape and size those of natural bone tissuespecifically trabecular bone They also noted that the 3Dpolyphosphazene scaffolds were able to promote osteoblastadhesion and proliferation throughout the entire 21-weekperiod of their study whereas adhesion to the 2D scaffoldswas not as effective Overall this study gave a good indicationthat polyphosphazene materials were suitable for bone tissueengineering Ambrosio et al [71] also investigated the appli-cability of polyphosphazenes to bone tissue repair throughthe development of polyphosphazene-hydroxyapatite com-posites They formed these composites (in a 1 3 ratio ofpolymerceramic) by dissolving the polymer in THF mixingwith hydroxyapatite particles vortexing the mixture andprecipitating the mixture with hexanes to form a putty-likematerial from which cylindrical samples were formed Thecomposite material interacted favorably withMC3T3-E1 cells(osteoblast-like cell line) and demonstrated improved celladhesion and proliferation in comparison to polystyrene-coated tissue culture plates (TCPS) They also showed thatthe composites had mechanical properties suitable to bonetissue engineering applications and that these properties weremaintained throughout the degradation process of the mate-rialThis study demonstrated the utility of polyphosphazenesas biomaterials for bone tissue engineering purposes Morerecently Morozowich et al [76] investigated the possibilityof incorporating antioxidants into polyphosphazene mate-rials to enhance their suitability as biomaterials for hardtissue engineering applications such as bone tissue Theywere capable of synthesizing ferulic acid an antioxidantand amino acid ester cosubstituted polyphosphazenes thatshowed degradation and UV-crosslinking properties suitablefor hard tissue engineering applications This suggested thematerialrsquos potential use for these applications although cyto-toxicity has yet to be fully investigated Although polyphosp-hazenes are thought to be osteoinductivematerials because oftheir phosphorus-containing feature they appeared to be lesssupportive to cell growth compared with the commonly usedaliphatic polyesters Muscle-inspired modification of fibrouspolyphosphazene mats with poly(dopamine) is reported toovercome this apparent limitation [77]

42 Nerve Tissue Engineering As alluded earlier Langoneet al [60] used polyphosphazene materials towards nervetissue engineering that investigated polymeric tubular nerveguides as prosthetics to promote nerve regeneration Theyshowed that poly[(ethyl alanato)

14(imidazolyl)

06phosp-

hazene] tubular constructs were capable of promoting thein vivo reattachment of experimentally transected rat sciaticnerves and that the new tissue was populated with cellssimilar to native neural tissues In a similar study [78]poly[bis(ethyl alanato)phosphazene] constructs were madefor neural tissue engineering by dipping a glass capillaryinto a polymer solution and allowing the solvent to evapo-rate leaving only the polymeric material This process wasrepeated until a polymer film of appropriate thickness wasformed at which point the glass capillary and polymercoating were dried and finally the glass capillary was removedfrom inside the polyphosphazene constructThese constructswere then implanted in vivo into Wistar rats which had theirright ischiatic nerve transected and the excised portion wasreplaced with the polymer conduit The polyphosphazeneconstructs remained implanted for time periods of 30 90and 180 days and were compared to control experimentswhere the excised area of the right nerve was replaced witha portion of the left ischiatic nerve Studies showed little tono toxicity of the absorbable polyphosphazene material aswell as nerve regeneration properties including myelinatedand unmyelinated nerve fibers similar to the control autol-ogous graft Overall this work showed the successful useof polyphosphazene-based materials for the development ofnerve guides in the regeneration of neural tissue

More recently Zhang et al [79] have studied polyphos-phazenes as conductive and degradable polymers for usein nerve tissue engineering Conductivity is an importantaspect of nerve tissue engineering considering that neuralsignals are propagated along nerve cells via electrical chargesand therefore the polymers that are used to regenerate thesetissues must be capable of transmitting waves of electricityA cosubstituted polyphosphazene material consisting of par-ent aniline pentamer (PAP) and glycine ethyl ester (GEE)was synthesized and formed into thin films for degrada-tion and biocompatibility testing The poly[(glycine ethylester)(aniline pentamer) phosphazene] (PGAP) polymer wasshown to have good electroactivity using cyclic voltammetrymeasurements meaning that the material would be suitablefor propagating neural signals Thin films of both PGAP andpoly[bis(glycine ethyl ester)phosphazene] (PGEE) materialswere subjected to degradation studies in PBS at 37∘C over astudy period of 70 days and showedmass losses of about 50and 70 respectively The mass loss of the PGAP was lessthan that of the PGEE due to the increased hydrophobicityof the aniline pentamer side chain and therefore decreasedrate of hydrolysis since the hydrophobic side chains stericallyhinder the approach of water towards the backbone Cellviability of the PGAP material was assessed using RSC96Schwann cells and was compared to a thin film of poly-DL-lactic acid (PDLLA) as a control since it has extensivelybeen shown to be biocompatible with numerous types ofcells RSC96 Schwann cells were chosen as Schwann cellsare integral parts in the peripheral nervous system not only


as supportive cells but also to help with the myelinationof the axons that propagate neural signals The studiesshowed improved cell adhesion to the PGAP material incomparison to PDLLA as well as no significant differencein cytotoxicity between the two materials Overall this workproves the usefulness of polyphosphazene-based materialsas conductive and biodegradable polymers for nerve tissueengineering applications

43 Tendon and Ligament Tissue Engineering In 2012Peach et al [80] analyzed polyphosphazene-functionalizedpoly(120576-caprolactone) (PCL) materials for their applicationin tendon tissue engineering In their studies electro-spun fibrous mats of PCL with average fiber diametersof 3000 plusmn 1700 nm coated with poly[(ethyl alanato)

1(p-

methyl phenoxy)1phosphazene] (PNEA-mPh) were used to

investigate cell behavior in response to the materials Humanmesenchymal stem cells (hMSC) were tested for their adhe-sion infiltration proliferation and differentiation propertieswhen exposed to the PNEA-mPh coated PCL constructsThe PNEA-mPh coated materials showed enhanced celladhesion and infiltration as compared to the uncoated PCLfibers due to the increased surface roughness created by thedip-coating process Cell proliferation was analyzed usingthe PicoGreen assay and showed that both materials werecapable of sustaining long-term growth of the hMSCs in vitroIn order for tissue engineering constructs to be clinicallyrelevant oftentimes the cells that comprise the tissue mustbe differentiated to the appropriate phenotype otherwise thetissuemay fail in vivo In the case of tissue-engineered tendongrafts the cells should undergo tenogenic differentiation byincreasing tenomodulin expression a late tendon differen-tiation marker protein Both the uncoated and coated PCLfibrous mats expressed scleraxis equally an early tendon dif-ferentiation marker protein but the polyphosphazene coatedmats showed increased tenomodulin expression indicatingthat this material was more phenotypically mature and abetter candidate as a tendon regeneration material thanthe uncoated counterpart The PNEA-mPh functionalizedmaterial also showed an increased ratio of collagen I tocollagen III as per real-time polymerase chain reaction (RT-PCR) analysis which also indicates the maturity of the differ-entiated cells into tendon cells Overall this study was ableto show the in vitro biocompatibility of polyphosphazene-coated materials towards human mesenchymal stem cellsas well as their ability to modulate appropriately the cellsrsquodifferentiation towards mature tendon cells

Polyphosphazene materials with improved elastomericproperties were studied by Nichol et al [81] in 2013 fortheir application in tendon and ligament tissue engineeringapplications For this study they investigated the influenceof changing alkyl ester chain lengths between five and eightcarbons on mechanical properties and degradation rates ofL-alanine and L-phenylalanine alkyl ester polyphosphazenematerials They determined that the glass transition temper-atures (T

119892) of the materials decreased with increasing alkyl

ester chain length due to increased flexibility of the alkyl sidechain and improved elastomeric properties of the polymer

It was also observed that the T119892rsquos of the phenylalanine mate-

rials were higher than those of the alanine counterpartswhichis likely due to the increased bulkiness and steric hindranceof the aromatic side chain that in turn increases the rigidity ofthe overall polymer For degradation studies square (5 cm times5 cm) solution-casted films were cut into 10mg samples andplaced in deionizedwater at pH63 and 37∘C for a time periodof 12 weeks After the specified weeks the aqueous media wastested for pH and the remaining polymer samplewasweighedand a GPC analysis was performed to determine mass lossand molecular weight decline respectively The resulting pHof the aqueous media varied between 52 and 68 Overall thephenylalanine-based materials showed decreased molecularweight decline in comparison to the alanine-based materialsindependent of the length of the alkyl ester side chainThis is most likely due to the increased steric hindrance ofthe backbone due to the large aromatic rings in the sidechain of phenylalanine which prevents water from reachingthe bonds that are to be hydrolyzed The phenylalaninematerials were also capable of forming better films whichmakes them less susceptible to hydrolysis Taken as a wholethe phenylalanine polyphosphazenes were shown to be themost suitable materials as scaffolds for soft tissue engineeringapplications due to their improved elastomeric properties andslow degradation rates

44 Polyphosphazenes for Drug Delivery Poly(organopho-sphazene)s were tested as delivery vehicles for the anti-cancer drug doxorubicin (DOX) [51] A polyphosphazenewith L-isoleucine ethyl ester (IleOEt) glycine glycine allylester (GlyGlyOAll) and 120572-amino-120596-methoxy-poly(ethyleneglycol) (AMPEG 550) substituents was synthesized andsubsequently conjugated with DOX through the pendantcarboxylic acid groups after removing the allyl protectinggroups on glycine glycine (poly[(IleOEt)

122(GlyGlyOH)

007

(GlyGlyODOX)005

(AMPE G550)066

phosphazene]) Thesematerials were shown to be injectable as a solution andprecipitate into a gel material upon heating which is suitablefor targeted drug delivery applications as it maintains thedrug in the desired location especially tumor sites Thematerial was tested in vitro for degradation properties drug(DOX) release profile and antitumor activity Degradationstudies and release profiles were performed in PBS (001MpH 74) at 37∘C over 30 days The mass loss after 30 dayswas approximately 60 and themolecular weight decline wasslightly less than 40TheDOX release profile demonstrateda sustained release of the drug which is ideal for most drugdelivery techniques The in vitro antitumor activity of theDOX-conjugated polyphosphazene material was comparedto both the polyphosphazene material alone and DOX aloneas controls using human breast cancer (MCF-7) and cervicalcarcinoma (HeLa) cell lines It was shown that the uncon-jugated polyphosphazene did not act as an antitumor agentwith either cell type with an inhibitory concentration (IC

50)

greater than 30 120583M The DOX-conjugated polyphosphazeneon the other hand showed IC

50similar to those of the DOX

alone for bothMCF-7 andHeLa cell lines with approximately1 120583M and 02 120583M respectively In vivo antitumor activity


1hr

4hr

1hr

1hr 3hr 6hr

7day

7day 28day

18day

14day

14day

21day

21day(a)

(b)

(c)

Figure 4 In vivo degradation process and in vivo localization of poly(organophosphazene)-doxorubicin (DOX) conjugate Biodegradationas a time-dependent mass loss of intratumorally injected polymer-DOX conjugate (a) Time-dependent fluorescence image of intratumorallyinjected DOX solution (30mgkg) (b) Time-dependent fluorescence image of intratumorally injected poly(organophosphazene)-DOXconjugate hydrogel (100mL equivalent to 223mgkg of DOX) (c) Reprinted from [51] with permission from Elsevier

analyses were performed on mice models that had beensubcutaneously implanted with tumor cells (SNU-601 humangastric cancer cell line) The mice were injected with twoconcentrations of the DOX-conjugated polyphosphazene asolution of free DOX and saline as a control The tumorvolume of the saline control steadily increased throughoutthe 28-day span of the study whereas the tumor volumesdecreased for all of the DOX containing solutions indicatinggrowth inhibition of the tumor (Figure 4) The free DOXsolution showed tumor suppression of about 62 by day 4followed by a slight increase in relative tumor volume at day 6and death of animal by day 12 due to the toxicity of high levelsof DOXThe polyphosphazene-DOX conjugates on the otherhand showed prolonged tumor suppression throughout theentire study period The higher dosage of DOX-conjugated(445mg of DOX per kg weight of mouse) material showedtumor suppression of 47 55 and 75 at 4 12 and 28days and was not so toxic as to kill the animal model unlikethe free DOX solution This study shows the great potentialof polyphosphazenematerials even over traditional methodssuch as bolus injections for sustained drug delivery and otherbiomedical applications that require gradually degradingbiomaterials

Recently Song and coworkers have been developingpoly(organophosphazene)s that are injectable and containanticancer agents such as silibinin [53] and camptothecin[82] They used L-isoleucine ethyl ester and deprotectedglycine glycine allyl ester substituents and were able toconjugate the drugs through the pendant carboxylic acidgroups Both studies investigated the in vitro degradationproperties and drug release profiles of the two conjugatedmaterials In both cases the drug showed sustained release

over the time frame of the study which is especially beneficialfor drugs that may be lethal at high concentrations andcannot therefore be administered as bolus injections Invitro and in vivo studies of antitumor activity were per-formed on both the silibinin- and camptothecin-conjugatedpolyphosphazene materials and both proved to have tumorinhibition effects against HT-29 colon cancer cell line Forthe in vivo analyses solutions of both the polymer-drugconjugate and drug only were injected into a site previouslyimplanted with an HT-29 cell xenograft and in all casesthe polymer-drug conjugates were just as effective at tumorinhibition as the drug alone but without the toxic sideeffects of the drug only solution In the silibinin-based studythe researchers also performed Western blot analyses anddetermined that silibinin elicited an antiangiogenic effect asobserved by the protein compliment being expressed by thecells Overall poly(organophosphazene)s conjugated withanticancer agents have shown to be successful as injectablethermosensitive hydrogels for targeted drug delivery

5 Polyphosphazene Blends as Biomaterials

Despite polyphosphazenes having inherent tunabilitythrough their side chains occasionally this is insufficient tomatch the required material properties of specific biomedicalapplications and thus polyphosphazene blends have alsobeen explored as potential biomaterials Lin et al [52]investigated the effect of varying polymer ratios on themorphology of electrospun mats of poly[(alanine ethylester)067

(glycine ethyl ester)033

phosphazene] (PAGP) andgelatin The polymer ratios tested were from 0 to 90 weight


percent (wt) gelatin to PAGP and these resulted in meanfiber diameters between 300 nm and 1 120583m Higher gelatincontent led to homogeneously distributed fibers with largerdiameter fibers At lower gelatin ratios (below 50wt)fibers showed a heterogeneous morphology with a gelatincore and PAGP shell Also the water contact angles of thematerials showed that the PAGP material is significantlymore hydrophobic than the gelatin and the overall surfacehydrophobicity of the material can be tailored by adjustingthe ratios of the two copolymers in the blend This tunabilityof surface hydrophobicity and fiber diameter by varyingratios of the copolymers in polyphosphazene blends mayfurther increase their utility in biomedical applications inthe future

Blends of polyphosphazenes andpolyesters as biomimeticscaffolds for bone regeneration have also been studied [83ndash85] For instance nanofibers of PLAGA poly[(glycylglycineethyl ester)

1(phenyl phenoxy)

1phosphazene] (PPHOS) and

blends of the two together were formed via electrospinningtechniques [83] The glycine dipeptide was incorporatedto minimize phase separation of the two polymers in theblend fibers by hydrogen bonding with PLAGA The largearomatic phenyl phenoxy groups were used to maintain themechanical properties such as compression resistance andhydrophobicity of the blend materials Nonwoven mats withfiber diameters between 50 and 500 nm had similar elasticmodulus and ultimate tensile strength to PLAGA indicatingtheir appropriate mechanical properties for bone tissue engi-neering applications In vitro these 3D biomimetic scaffoldswere capable of promoting cell infiltration as indicated bythe migration of cells from the blend layers to the interlayerspace and extracellular matrix deposition by the osteoblastcells as shown by the phenotypemarker expression includingECM proteins such as osteopontin Overall this study showsthe success of polyphosphazene blend materials as potentialbiomaterials for biomedical applications such as bone tissueengineering

6 Conclusions and Future Outlook

Throughout this review paper the potential of polyphosp-hazenes for use in biomedical applications has been exploredRather than focusing on the applications alone this reviewattempted to provide a larger overview of synthesis tech-niques and in-depth rationale of polyphosphazene poly-mers as biomaterials specifically their biocompatibilitybiodegradability and mechanical properties all of whichare key characteristics of biomaterials Polyphosphazenes arecurrently being extensively studied as scaffold materials anddrug delivery devices although their utility in other biomedi-cal applications have not yet been fully investigated As inter-est in the area of biocompatible poly(organo)phosphazenesgrows it is expected that these materials will be employedfor other tissue engineering applications such as tendon andblood vessel engineering as well as a wide range of otherbiomedical applications

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

The authors acknowledge the financial support providedby the Natural Sciences and Engineering Research CouncilCanada (NSERC)

References

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[2] J P Vacanti ldquoTissue engineering and the road to whole organsrdquoBritish Journal of Surgery vol 99 no 4 pp 451ndash453 2012

[3] C T Laurencin A M A Ambrosio M D Borden and JA Cooper Jr ldquoTissue engineering orthopedic applicationsrdquoAnnual Review of Biomedical Engineering vol 1 pp 19ndash46 1999

[4] P X Ma ldquoBiomimetic materials for tissue engineeringrdquoAdvanced Drug Delivery Reviews vol 60 no 2 pp 184ndash1982008

[5] M Deng S G Kumbar Y Wan U S Toti H R Allcockand C T Laurencin ldquoPolyphosphazene polymers for tissueengineering an analysis of material synthesis characterizationand applicationsrdquo Soft Matter vol 6 no 14 pp 3119ndash3132 2010

[6] R Langer ldquoEditorial tissue engineering perspectives chal-lenges and future directionsrdquo Tissue Engineering vol 13 no 1pp 1ndash2 2007

[7] J A Hubbell ldquoBiomaterials in tissue engineeringrdquo Biotechnol-ogy vol 13 no 6 pp 565ndash576 1995

[8] R S Langer and N A Peppas ldquoPresent and future applicationsof biomaterials in controlled drug delivery systemsrdquo Biomateri-als vol 2 no 4 pp 201ndash214 1981

[9] E S Place N D Evans and M M Stevens ldquoComplexity inbiomaterials for tissue engineeringrdquoNatureMaterials vol 8 no6 pp 457ndash470 2009

[10] L S Nair and C T Laurencin ldquoBiodegradable polymers asbiomaterialsrdquo Progress in Polymer Science vol 32 no 8-9 pp762ndash798 2007

[11] S G Kumbar K S Soppimath and T M AminabhavildquoSynthesis and characterization of polyacrylamide-grafted chi-tosan hydrogel microspheres for the controlled release ofindomethacinrdquo Journal of Applied Polymer Science vol 87 no9 pp 1525ndash1536 2003

[12] K A Athanasiou G G Niederauer and C M Agrawal ldquoSter-ilization toxicity biocompatibility and clinical applications ofpolylactic acidpolyglycolic acid copolymersrdquo Biomaterials vol17 no 2 pp 93ndash102 1996

[13] S J De Jong E R Arias D T S Rijkers C F Van Nostrum J JKettenes-Van denBosch andW EHennink ldquoNew insights intothe hydrolytic degradation of poly(lactic acid) participation ofthe alcohol terminusrdquo Polymer vol 42 no 7 pp 2795ndash28022001

[14] M S Taylor A U Daniels K P Andriano and J Heller ldquoSixbioabsorbable polymers In vitro acute toxicity of accumulateddegradation productsrdquo Journal of applied biomaterials vol 5 no2 pp 151ndash157 1994


[15] C J Bettinger ldquoSynthetic biodegradable elastomers for drugdelivery and tissue engineeringrdquo Pure and Applied Chemistryvol 83 no 1 pp 9ndash24 2011

[16] D Puppi F Chiellini A M Piras and E Chiellini ldquoPolymericmaterials for bone and cartilage repairrdquo Progress in PolymerScience vol 35 no 4 pp 403ndash440 2010

[17] H R Allcock ldquoRecent advances in phosphazene (phosphoni-trilic) chemistryrdquo Chemical Reviews vol 72 no 4 pp 315ndash3561972

[18] H R Allcock Chemistry and Applications of PolyphosphazenesWiley Interscience Hoboken NJ USA 2003

[19] H R Allcock R L Kugel and E G Stroh ldquoPhospho-nitrilic compounds XIII The structure and properties ofpoly(difluorophosphazene)rdquo Inorganic Chemistry vol 11 no 5pp 1120ndash1123 1972

[20] H R Allcock T J Fuller D P Mack K Matsumuraand K M Smeltz ldquoSynthesis of poly[(amino acid alkylester)phosphazenes]rdquo Macromolecules vol 10 no 4 pp 824ndash830 1977

[21] A K AndrianovPolyphosphazenes For Biomedical ApplicationsJohn Wiley amp Sons Hoboken NJ USA 2009

[22] J H L Crommen E H Schacht and E H G MenseldquoBiodegradable polymers II Degradation characteristics ofhydrolysis-sensitive poly[(organo)phosphazenes]rdquo Biomateri-als vol 13 no 9 pp 601ndash611 1992

[23] A Singh N R Krogman S Sethuraman et al ldquoEffect of sidegroup chemistry on the properties of biodegradable l-alaninecosubstituted polyphosphazenesrdquoBiomacromolecules vol 7 no3 pp 914ndash918 2006

[24] K J L Burg S Porter and J F Kellam ldquoBiomaterial develop-ments for bone tissue engineeringrdquo Biomaterials vol 21 no 23pp 2347ndash2359 2000

[25] R M Nerem and D Seliktar ldquoVascular tissue engineeringrdquoAnnual Review of Biomedical Engineering vol 3 pp 225ndash2432001

[26] H R Allcock ldquoThe synthesis of functional polyphosphazenesand their surfacesrdquo Applied Organometallic Chemistry vol 12no 10-11 pp 659ndash666 1998

[27] H R Allcock and R L Kugel ldquoSynthesis of high polymericalkoxy-and aryloxyphosphonitrilesrdquo Journal of the AmericanChemical Society vol 87 no 18 pp 4216ndash4217 1965

[28] A N Mujumdar S G Young R L Merker and J H MagillldquoA study of solution polymerization of polyphosphazenesrdquoMacromolecules vol 23 no 1 pp 14ndash21 1990

[29] H R Allcock C A Crane C T Morrissey et al ldquolsquoLivingrsquocationic polymerization of phosphoranimines as an ambienttemperature route to polyphosphazenes with controlled molec-ular weightsrdquo Macromolecules vol 29 no 24 pp 7740ndash77471996

[30] H R Allcock J M Nelson S D Reeves C H Honey-man and I Manners ldquoAmbient-temperature direct synthesisof poly(organophosphazenes) via the ldquolivingrdquo cationic poly-merization of organo-substituted phosphoraniminesrdquo Macro-molecules vol 30 no 1 pp 50ndash56 1997

[31] H R Allcock S D Reeves C R De Denus and C ACrane ldquoInfluence of reaction parameters on the living cationicpolymerization of phosphoranimines to polyphosphazenesrdquoMacromolecules vol 34 no 4 pp 748ndash754 2001

[32] C H Honeyman I Manners C T Morrissey and H RAllcock ldquoAmbient temperature synthesis of poly(dichloro-phosphazene) with molecular weight controlrdquo Journal of theAmerican Chemical Society vol 117 no 26 pp 7035ndash7036 1995

[33] E S Peterson T A Luther M K Harrup et al ldquoOn thecontributions to the materials science aspects of phosphazenechemistry by Professor Christopher W Allen the one-potsynthesis of linear polyphosphazenesrdquo Journal of Inorganic andOrganometallic Polymers and Materials vol 17 no 2 pp 361ndash366 2007

[34] S-K Kwon ldquoSynthesis of water-soluble methoxyethoxy-aminoarlyoxy cosubstituted polyphosphazenes as carriermolecules for bioactive agentsrdquo Bulletin of the Korean ChemicalSociety vol 21 no 10 pp 969ndash972 2000

[35] L E Freed G Vunjak-Novakovic R J Biron et al ldquoBiodegrad-able polymer scaffolds for tissue engineeringrdquo Biotechnologyvol 12 no 7 pp 689ndash693 1994

[36] S Yang K-F Leong Z Du and C-K Chua ldquoThe designof scaffolds for use in tissue engineering Part I Traditionalfactorsrdquo Tissue Engineering vol 7 no 6 pp 679ndash689 2001

[37] H R Allcock S R Pucher and A G ScopelianosldquoPoly[(amino acid ester)phosphazenes] synthesis crystallinityand hydrolytic sensitivity in solution and the solid staterdquoMacromolecules vol 27 no 5 pp 1071ndash1075 1994

[38] H RAllcock S R Pucher andAG Scopelianos ldquoPoly[(aminoacid ester)phosphazenes] as substrates for the controlled releaseof small moleculesrdquo Biomaterials vol 15 no 8 pp 563ndash5691994

[39] A K Andrianov A Marin and P Peterson ldquoWater-solublebiodegradable polyphosphazenes containing N-ethylpyrroli-done groupsrdquo Macromolecules vol 38 no 19 pp 7972ndash79762005

[40] J Crommen J Vandorpe and E Schacht ldquoDegradable poly-phosphazenes for biomedical applicationsrdquo Journal of Con-trolled Release vol 24 no 1ndash3 pp 167ndash180 1993

[41] J H L Crommen and E H Schacht ldquoSynthesis and evaluationof the hydrolytical stability of ethyl 2-(120572-amino acid)glycolatesand ethyl 2-(120572-amino acid)lactatesrdquo Bulletin Des Societes Chim-iques Belges vol 100 no 10 pp 747ndash758 1991

[42] S Lakshmi D S Katti and C T Laurencin ldquoBiodegradablepolyphosphazenes for drug delivery applicationsrdquo AdvancedDrug Delivery Reviews vol 55 no 4 pp 467ndash482 2003

[43] C T Laurencin C D Morris H Pierres-Jacques E RSchwartz A R Keaton and L Zou ldquoThe development of bonebioerodible polymer composites for skeletal tissue regenerationstudies of initial cell attachment and spreadrdquo Polymers ForAdvanced Technologies no 3 pp 369ndash364 1992

[44] L S Nair D A Lee J D Bender et al ldquoSynthesis characteriza-tion and osteocompatibility evaluation of novel alanine-basedpolyphosphazenesrdquo Journal of Biomedical Materials Research Avol 76 no 1 pp 206ndash213 2006

[45] L YQiu andK J Zhu ldquoNovel biodegradable polyphosphazenescontaining glycine ethyl ester and benzyl ester of amino acethy-droxamic acid as cosubstituents syntheses characterizationand degradation propertiesrdquo Journal of Applied Polymer Sciencevol 77 no 13 pp 2987ndash2995 2000

[46] E Schacht J Vandorpe S Dejardin Y Lemmouchi and LSeymour ldquoBiomedical applications of degradable polyphosp-hazenesrdquo Biotechnology and Bioengineering vol 52 no 1 pp102ndash108 1996

[47] S Sethuraman L S Nair S El-Amin et al ldquoIn vivo biodegrad-ability and biocompatibility evaluation of novel alanine esterbased polyphosphazenes in a rat modelrdquo Journal of BiomedicalMaterials Research A vol 77 no 4 pp 679ndash687 2006

[48] J Chlupac E Filova and L Bacakova ldquoBlood vesselreplacement 50 years of development and tissue engineering


paradigms in vascular surgeryrdquo Physiological researchAcademia Scientiarum Bohemoslovaca vol 58 pp S119ndashS139 2009

[49] H R Allcock and N L Morozowich ldquoBioerodible polyphosp-hazenes and their medical potentialrdquo Polymer Chemistry vol 3no 3 pp 578ndash590 2012

[50] Y J Lin Q H Deng and R G Jin ldquoEffects of processingvariables on the morphology and diameter of electrospunpoly(amino acid ester)phosphazene nanofibersrdquo Journal ofWuhan University of Technology-Materials Science vol 27 no2 pp 207ndash211 2012

[51] C Chun S M Lee C W Kim et al ldquoDoxorubicin-polyphosphazene conjugate hydrogels for locally controlleddelivery of cancer therapeuticsrdquo Biomaterials vol 30 no 27 pp4752ndash4762 2009

[52] Y-J Lin Q Cai L Li Q-F Li X-P Yang and R-G Jin ldquoCo-electrospun composite nanofibers of blends of poly[(amino acidester)phosphazene] and gelatinrdquo Polymer International vol 59no 5 pp 610ndash616 2010

[53] J-K Cho JW Park and S-C Song ldquoInjectable and biodegrad-able poly(organophosphazene) gel containing silibinin itsphysicochemical properties and anticancer activityrdquo Journal ofPharmaceutical Sciences vol 101 no 7 pp 2382ndash2391 2012

[54] S Wilfert A Iturmendi W Schoefberger et al ldquoWater-solublebiocompatible polyphosphazenes with controllable and pH-promoted degradation behaviorrdquo Journal of Polymer Science APolymer Chemistry vol 52 no 2 pp 287ndash294 2014

[55] Y Bi X Gong F He et al ldquoPolyphosphazenes containinglactic acid ester and methoxyethoxyethoxy side groupsmdashthermosensitive properties and in vitro degradation and bio-compatibilityrdquo Canadian Journal of Chemistry vol 89 no 10pp 1249ndash1256 2011

[56] C T Laurencin M E Norman H M Elgendy et al ldquoUse ofpolyphosphazenes for skeletal tissue regenerationrdquo Journal ofBiomedical Materials Research vol 27 no 7 pp 963ndash973 1993

[57] M Gumusderelioglu and A Gur ldquoSynthesis characterizationIn vitro degradation and cytotoxicity of poly[bis(ethyl 4-aminobutyro)phosphazene]rdquoReactive andFunctional Polymersvol 52 no 2 pp 71ndash80 2002

[58] C Armstrong and J F Staples ldquoThe role of succinate dehy-drogenase and oxaloacetate in metabolic suppression duringhibernation and arousalrdquo Journal of Comparative Physiology BBiochemical Systemic and Environmental Physiology vol 180no 5 pp 775ndash783 2010

[59] P Carampin M T Conconi S Lora et al ldquoElectrospunpolyphosphazene nanofibers for In vitro rat endothelial cellsproliferationrdquo Journal of Biomedical Materials Research A vol80 no 3 pp 661ndash668 2007

[60] F Langone S Lora F M Veronese et al ldquoPeripheral nerverepair using a poly(organo)phosphazene tubular prosthesisrdquoBiomaterials vol 16 no 5 pp 347ndash353 1995

[61] Y Fan M Kobayashi and H Kise ldquoSynthesis and biodegra-dation of Poly (ester amide)s containing amino acid residuesthe effect of the stereoisomeric composition of L- and D-Phenylalanines on the enzymatic degradation of the polymersrdquoJournal of Polymer Science A Polymer Chemistry vol 40 no 3pp 385ndash392 2002

[62] P X Ma lsquoTissue Engineeringrsquo Encyclopedia of Polymer Scienceand Technology John Wiley amp Sons Hoboken NJ USA 2004

[63] J-K Cho K-Y Hong J W Park H-K Yang and S-CSong ldquoInjectable delivery system of 2-methoxyestradiol for

breast cancer therapy using biodegradable thermosensitivepoly(organophosphazene) hydrogelrdquo Journal of Drug Targetingvol 19 no 4 pp 270ndash280 2011

[64] G D Kang S H Cheon G Khang and S-C Song ldquoTher-mosensitive poly(organophosphazene) hydrogels for a con-trolled drug deliveryrdquo European Journal of Pharmaceutics andBiopharmaceutics vol 63 no 3 pp 340ndash346 2006

[65] B H Lee and S-C Song ldquoSynthesis and characterization ofbiodegradable thermosensitive poly(organophosphazene) gelsrdquoMacromolecules vol 37 no 12 pp 4533ndash4537 2004

[66] G H Altman R L Horan H H Lu et al ldquoSilkmatrix for tissueengineered anterior cruciate ligamentsrdquo Biomaterials vol 23no 20 pp 4131ndash4141 2002

[67] K S Katti ldquoBiomaterials in total joint replacementrdquo Colloidsand Surfaces B Biointerfaces vol 39 no 3 pp 133ndash142 2004

[68] G Konig T N McAllister N Dusserre et al ldquoMechanicalproperties of completely autologous human tissue engineeredblood vessels compared to human saphenous vein and mam-mary arteryrdquo Biomaterials vol 30 no 8 pp 1542ndash1550 2009

[69] S SarkarH J Salacinski GHamilton andAM Seifalian ldquoThemechanical properties of infrainguinal vascular bypass graftstheir role in influencing patencyrdquo European Journal of Vascularand Endovascular Surgery vol 31 no 6 pp 627ndash636 2006

[70] S Sethuraman L S Nair S El-Amin et al ldquoMechanical prop-erties and osteocompatibility of novel biodegradable alaninebased polyphosphazenes side group effectsrdquo Acta Biomateri-alia vol 6 no 6 pp 1931ndash1937 2010

[71] A M A Ambrosio J S Sahota C Runge et al ldquoNovelpolyphosphazene-hydroxyapatite composites as biomaterialsrdquoIEEE Engineering inMedicine and BiologyMagazine vol 22 no5 pp 18ndash26 2003

[72] J L Brown L S Nair and C T Laurencin ldquoSolventnon-solvent sintering a novel route to create porous microspherescaffolds for tissue regenerationrdquo Journal of Biomedical Materi-als Research B Applied Biomaterials vol 86 no 2 pp 396ndash4062008

[73] C T Laurencin S F ElAmin S E Ibim et al ldquoA highly porous3-dimensional polyphosphazene polymer matrix for skeletaltissue regenerationrdquo Journal of Biomedical Materials Researchvol 30 no 2 pp 133ndash138 1996

[74] S P Nukavarapu S G Kumbar J L Brown et al ldquoPolyphosp-hazene nano-hydroxyapatite composite microsphere scaffoldsfor bone tissue engineeringrdquoBiomacromolecules vol 9 no 7 pp1818ndash1825 2008

[75] L S Nair S Bhattacharyya J D Bender et al ldquoFabrication andoptimization of methylphenoxy substituted polyphosphazenenanofibers for biomedical applicationsrdquo Biomacromoleculesvol 5 no 6 pp 2212ndash2220 2004

[76] N L Morozowich J L Nichol R J Mondschein and H RAllcock ldquoDesign and examination of an antioxidant-containingpolyphosphazene scaffold for tissue engineeringrdquo PolymerChemistry vol 3 no 3 pp 778ndash786 2012

[77] Y Li Y Shi S Duan et al ldquoElectrospun biodegradablepolyorganophosphazene fibrous matrix with poly(dopamine)coating for bone regenerationrdquo Journal of Biomedical MaterialsResearch A Early View-Online Version of Record PublishedBefore Inclusion in An Issue 2013

[78] N N Aldini M Fini M Rocca et al ldquoPeripheral nervereconstruction with bioabsorbable polyphosphazene conduitsrdquoJournal of Bioactive and Compatible Polymers vol 12 no 1 pp3ndash13 1997


[79] Q Zhang Y Yan S Li and T Feng ldquoThe synthesis andcharacterization of a novel biodegradable and electroactivepolyphosphazene for nerve regenerationrdquoMaterials Science andEngineering C vol 30 no 1 pp 160ndash166 2010

[80] M S Peach R James U S Toti et al ldquoPolyphosphazenefunctionalized polyester fiber matrices for tendon tissue engi-neering In vitro evaluation with human mesenchymal stemcellsrdquo Biomedical Materials vol 7 no 4 pp 1ndash13 2012

[81] J L Nichol N L Morozowich and H R Allcock ldquoBiodegrad-able alanine and phenylalanine alkyl ester polyphosphazenesas potential ligament and tendon tissue scaffoldsrdquo PolymerChemistry vol 4 no 3 pp 600ndash606 2013

[82] J K Cho C Chun H J Kuh and S C Song ldquoInjectablepoly(organophosphazene)-camptothecin conjugate hydrogelssynthesis characterization and antitumor activitiesrdquo EuropeanJournal of Pharmaceutics and Biopharmaceutics vol 81 no 3pp 582ndash590 2012

[83] M Deng S G Kumbar L S Nair A L Weikel H RAllcock and C T Laurencin ldquoBiomimetic structures bio-logical implications of dipeptide-substituted polyphosphazene-polyester blend nanofiber matrices for load-bearing boneregenerationrdquoAdvanced FunctionalMaterials vol 21 no 14 pp2641ndash2651 2011

[84] N R Krogman A Singh L S Nair C T Laurencin andH R Allcock ldquoMiscibility of bioerodible polyphosphazenepoly(lactide-co-glycolide) blendsrdquo Biomacromolecules vol 8no 4 pp 1306ndash1312 2007

[85] A L Weikel S G Owens N L Morozowich et al ldquoMisci-bility of choline-substituted polyphosphazenes with PLGA andosteoblast activity on resulting blendsrdquo Biomaterials vol 31 no33 pp 8507ndash8515 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


an alternative to natural materials Biodegradability is adesirable feature of a biomaterial used in tissue engineeringsince the goal is that it acts as a temporary scaffold holdingthe growing tissue in place until the natural extracellularmatrix has sufficiently developed Beyond that point the scaf-fold should breakdown into nontoxic degradation productscapable of being disposed of by the body leaving only thenewly formed tissue There are a wide variety of syntheticbiodegradable polymers that have been and continue to beexplored including polyesters polyanhydrides polyacetalsand poly(120572-amino acids) [10] Despite their improvementover natural polymers with regard to degradation andmechanical properties synthetic polymers have their ownlimitations A common problem of synthetic polymers suchas poly(lactic acid) (PLA) is the formation of acidic productsduring the degradation process which leads to diminishedmechanical strength of the material and compromised cellfunction in the acidic environment [12ndash14] The quest forbiomaterials with tunable degradation rates and mechanicalproperties which also maintain cell function and lack theformation of toxic degradation products is an active area ofresearch [15 16]

Sustained research towards new biomaterials for tissueengineering and regenerative medicine applications has ledto the utilization of polyphosphazenes as a class of novelmaterials Polyphosphazenes are comprised of an inorganicbackbone of repeating phosphorus and nitrogen atoms withalternating single and double bonds (Figure 1 Structure 1c)[6 17ndash19] Extending from each of the phosphorus atoms aretwo organic side chains which can range from alkoxy andaryloxy substituents to amino acids giving a large variety ofpotential polymers [5 18 20 21] Changing the organic sidegroups and their ratios if multiple different side groups areattached to the same polymer backbone allows substantialtunability of the physical and degradation properties ofthe material [18 22 23] Therefore altering the organicsubstituents can be quite useful in tailoring the mechanicalproperties and degradation rates of the biomaterial to suitthe desired tissue engineering application such as bone tissueor blood vessels which require drastically different physicalproperties [24 25]

2 Synthesis of Polyphosphazenes

The synthesis of polyphosphazenes such as those shown inFigure 1 is typically via a two-step reaction beginning withthe thermal ring opening polymerization of hexachloro-cyclotriphosphazene (1a) the cyclic trimer to the linearpoly(dichlorophosphazene) (1b) precursor Next the organicside chains are attached to the polymer backbone througha nucleophilic macromolecular substitution of the organicsubstituents for the phosphorus-bound chlorine atoms [1821 26] The following two sections succinctly will describethe individual steps of polyorganophosphazene synthesisshowing the vast range of materials that can be generated

21 Thermal Ring Opening Polymerization-Bulk PhaseAlthough the thermal ring polymerization of the trimer (1a)

CH3

CH3

N

N

N

N

N

P

P

P

P

P

O

O

O

OO

O

O

O

O

O

O

O

HO

HOHO

HO

H

H

HH

H

HH

H HH

R

R

R

R

OR998400

OR998400

OR998400

NH

NH

NH2

NH2

OH

OH

OH

OH

2

3

451c

R998400O

n

n

n

n

n

Figure 1 Structures of various polyphosphazenes includingsteroidal substituents (2) carbohydrates (3) amino acid esters(4) and side chain-bound amino acid esters (5) to name a fewAdapted from [5] by permission of the Royal Society of Chemistry(httpdxdoiorg101039B926402G)

to linear poly(dichlorophosphazene) was attempted inthe late 1800s by H N Stokes a useful material thatwas soluble and capable of being functionalized was notrealized until the 1960s The initial thermal ring openingpolymerization performed by Stokes lead to a product thatwas insoluble due to crosslinking and that was readilysusceptible to hydrolysis when exposed to moisture [18]In 1965 Allcock and Kugel [27] were able to synthesislinear poly(dichlorophosphazene) through a well-controlledthermal ring opening polymerization from the cyclic trimerhexachlorocyclotriphosphazene according to Scheme 1 Theproduct obtained was soluble allowing it to be modifiedfurther by macromolecular substitution of the reactivePndashCl bonds with organic and organometallic nucleophilesThe thermal ring opening polymerization techniquedeveloped by Allcock et al is the most commonly usedroute to prepare the linear poly(dichlorophosphazene)precursor [5 18] A typical process involves reactingpurified hexachlorocyclotriphosphazene trimer at 250∘Cover 5 days in an evacuated polymerization tube Atthis point soluble poly(dichlorophosphazene) has beenformed that can be purified and functionalized via


P

P P

P P P P

PN

N N

N N N

N

N

ClCl

Cl

Cl

Cl Cl

ClCl

250∘C 120h

NHR

NHR

NR2

NR2

HN

R 2

MR

OR

OR

1a 1b

H 2NR

R

R

NaOR

n

n n n n












(a) (b)

10 120583m 10 120583m


100

80

60

40

20

0

0 5 10 15 20 25

Time (day)

Cum

ulat

ive a

mou

nt o

frel

ease

d sil

ibin

in (




(c)


PN

R

R


0 sRoom temperature

Body temperature

10 s 30 s 60 s

0 s 10 s 30 s 60 s

n








1





14





1(p-



1(p-phenyl phenoxy)

1phosphazene]



14(imidazolyl)










1(ethyl




P

P

P

NH

NH

NH

HN

H

N

N

O

O

O

O

O

O

O

O

O

O

O

O

O

O

R

R

R

R

N N

NN

N

N

N

N

P

P

P

P

minus OHH2O

H2O

H2O

H2O

H2OH2O

H2O

H2N

Phosphazanes

PhosphazanesP

P

P

P

NH

R

R

R

OH

OH

OH



minus

H2Nminus

H2Nminus

∙ ∙

∙ ∙

∙ ∙ ∙ ∙

∙ ∙

∙ ∙

∙ ∙

∙∙

∙∙

∙∙

∙∙

∙∙

∙∙

∙ ∙

Hydroxyphosphazene

1

2

3

R998400

R998400

R998400

R998400

R998400

R998400







Glycine

Methyl 35 days [37]

Ethyl




Alanine

Methyl 5 weeks [37]











Com

pres

sive s

treng

th (M

Pa)

PNEA PNEA50 mPh50

lowast


100

80

60

40

20

0


50

mPh50


80

PhPh20


50

PhPh50










14(imidazolyl)

06phosp-






1(p-









122(GlyGlyOH)

007

(GlyGlyODOX)005

(AMPE G550)066


50)





1hr

4hr

1hr

1hr 3hr 6hr

7day

7day 28day

18day

14day

14day

21day

21day(a)

(b)

(c)












1(phenyl phenoxy)







Acknowledgment


References
































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















P

P P

P P P P

PN

N N

N N N

N

N

ClCl

Cl

Cl

Cl Cl

ClCl

250∘C 120h

NHR

NHR

NR2

NR2

HN

R 2

MR

OR

OR

1a 1b

H 2NR

R

R

NaOR

n

n n n n












(a) (b)

10 120583m 10 120583m


100

80

60

40

20

0

0 5 10 15 20 25

Time (day)

Cum

ulat

ive a

mou

nt o

frel

ease

d sil

ibin

in (




(c)


PN

R

R


0 sRoom temperature

Body temperature

10 s 30 s 60 s

0 s 10 s 30 s 60 s

n








1





14





1(p-



1(p-phenyl phenoxy)

1phosphazene]



14(imidazolyl)










1(ethyl




P

P

P

NH

NH

NH

HN

H

N

N

O

O

O

O

O

O

O

O

O

O

O

O

O

O

R

R

R

R

N N

NN

N

N

N

N

P

P

P

P

minus OHH2O

H2O

H2O

H2O

H2OH2O

H2O

H2N

Phosphazanes

PhosphazanesP

P

P

P

NH

R

R

R

OH

OH

OH



minus

H2Nminus

H2Nminus

∙ ∙

∙ ∙

∙ ∙ ∙ ∙

∙ ∙

∙ ∙

∙ ∙

∙∙

∙∙

∙∙

∙∙

∙∙

∙∙

∙ ∙

Hydroxyphosphazene

1

2

3

R998400

R998400

R998400

R998400

R998400

R998400







Glycine

Methyl 35 days [37]

Ethyl




Alanine

Methyl 5 weeks [37]











Com

pres

sive s

treng

th (M

Pa)

PNEA PNEA50 mPh50

lowast


100

80

60

40

20

0


50

mPh50


80

PhPh20


50

PhPh50










14(imidazolyl)

06phosp-






1(p-









122(GlyGlyOH)

007

(GlyGlyODOX)005

(AMPE G550)066


50)





1hr

4hr

1hr

1hr 3hr 6hr

7day

7day 28day

18day

14day

14day

21day

21day(a)

(b)

(c)












1(phenyl phenoxy)







Acknowledgment


References
































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b)

10 120583m 10 120583m


100

80

60

40

20

0

0 5 10 15 20 25

Time (day)

Cum

ulat

ive a

mou

nt o

frel

ease

d sil

ibin

in (




(c)


PN

R

R


0 sRoom temperature

Body temperature

10 s 30 s 60 s

0 s 10 s 30 s 60 s

n








1





14





1(p-



1(p-phenyl phenoxy)

1phosphazene]



14(imidazolyl)










1(ethyl




P

P

P

NH

NH

NH

HN

H

N

N

O

O

O

O

O

O

O

O

O

O

O

O

O

O

R

R

R

R

N N

NN

N

N

N

N

P

P

P

P

minus OHH2O

H2O

H2O

H2O

H2OH2O

H2O

H2N

Phosphazanes

PhosphazanesP

P

P

P

NH

R

R

R

OH

OH

OH



minus

H2Nminus

H2Nminus

∙ ∙

∙ ∙

∙ ∙ ∙ ∙

∙ ∙

∙ ∙

∙ ∙

∙∙

∙∙

∙∙

∙∙

∙∙

∙∙

∙ ∙

Hydroxyphosphazene

1

2

3

R998400

R998400

R998400

R998400

R998400

R998400







Glycine

Methyl 35 days [37]

Ethyl




Alanine

Methyl 5 weeks [37]











Com

pres

sive s

treng

th (M

Pa)

PNEA PNEA50 mPh50

lowast


100

80

60

40

20

0


50

mPh50


80

PhPh20


50

PhPh50










14(imidazolyl)

06phosp-






1(p-









122(GlyGlyOH)

007

(GlyGlyODOX)005

(AMPE G550)066


50)





1hr

4hr

1hr

1hr 3hr 6hr

7day

7day 28day

18day

14day

14day

21day

21day(a)

(b)

(c)












1(phenyl phenoxy)







Acknowledgment


References
































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















1





14





1(p-



1(p-phenyl phenoxy)

1phosphazene]



14(imidazolyl)










1(ethyl




P

P

P

NH

NH

NH

HN

H

N

N

O

O

O

O

O

O

O

O

O

O

O

O

O

O

R

R

R

R

N N

NN

N

N

N

N

P

P

P

P

minus OHH2O

H2O

H2O

H2O

H2OH2O

H2O

H2N

Phosphazanes

PhosphazanesP

P

P

P

NH

R

R

R

OH

OH

OH



minus

H2Nminus

H2Nminus

∙ ∙

∙ ∙

∙ ∙ ∙ ∙

∙ ∙

∙ ∙

∙ ∙

∙∙

∙∙

∙∙

∙∙

∙∙

∙∙

∙ ∙

Hydroxyphosphazene

1

2

3

R998400

R998400

R998400

R998400

R998400

R998400







Glycine

Methyl 35 days [37]

Ethyl




Alanine

Methyl 5 weeks [37]











Com

pres

sive s

treng

th (M

Pa)

PNEA PNEA50 mPh50

lowast


100

80

60

40

20

0


50

mPh50


80

PhPh20


50

PhPh50










14(imidazolyl)

06phosp-






1(p-









122(GlyGlyOH)

007

(GlyGlyODOX)005

(AMPE G550)066


50)





1hr

4hr

1hr

1hr 3hr 6hr

7day

7day 28day

18day

14day

14day

21day

21day(a)

(b)

(c)












1(phenyl phenoxy)







Acknowledgment


References
































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























1(ethyl




P

P

P

NH

NH

NH

HN

H

N

N

O

O

O

O

O

O

O

O

O

O

O

O

O

O

R

R

R

R

N N

NN

N

N

N

N

P

P

P

P

minus OHH2O

H2O

H2O

H2O

H2OH2O

H2O

H2N

Phosphazanes

PhosphazanesP

P

P

P

NH

R

R

R

OH

OH

OH



minus

H2Nminus

H2Nminus

∙ ∙

∙ ∙

∙ ∙ ∙ ∙

∙ ∙

∙ ∙

∙ ∙

∙∙

∙∙

∙∙

∙∙

∙∙

∙∙

∙ ∙

Hydroxyphosphazene

1

2

3

R998400

R998400

R998400

R998400

R998400

R998400







Glycine

Methyl 35 days [37]

Ethyl




Alanine

Methyl 5 weeks [37]











Com

pres

sive s

treng

th (M

Pa)

PNEA PNEA50 mPh50

lowast


100

80

60

40

20

0


50

mPh50


80

PhPh20


50

PhPh50










14(imidazolyl)

06phosp-






1(p-









122(GlyGlyOH)

007

(GlyGlyODOX)005

(AMPE G550)066


50)





1hr

4hr

1hr

1hr 3hr 6hr

7day

7day 28day

18day

14day

14day

21day

21day(a)

(b)

(c)












1(phenyl phenoxy)







Acknowledgment


References
































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















P

P

P

NH

NH

NH

HN

H

N

N

O

O

O

O

O

O

O

O

O

O

O

O

O

O

R

R

R

R

N N

NN

N

N

N

N

P

P

P

P

minus OHH2O

H2O

H2O

H2O

H2OH2O

H2O

H2N

Phosphazanes

PhosphazanesP

P

P

P

NH

R

R

R

OH

OH

OH



minus

H2Nminus

H2Nminus

∙ ∙

∙ ∙

∙ ∙ ∙ ∙

∙ ∙

∙ ∙

∙ ∙

∙∙

∙∙

∙∙

∙∙

∙∙

∙∙

∙ ∙

Hydroxyphosphazene

1

2

3

R998400

R998400

R998400

R998400

R998400

R998400







Glycine

Methyl 35 days [37]

Ethyl




Alanine

Methyl 5 weeks [37]











Com

pres

sive s

treng

th (M

Pa)

PNEA PNEA50 mPh50

lowast


100

80

60

40

20

0


50

mPh50


80

PhPh20


50

PhPh50










14(imidazolyl)

06phosp-






1(p-









122(GlyGlyOH)

007

(GlyGlyODOX)005

(AMPE G550)066


50)





1hr

4hr

1hr

1hr 3hr 6hr

7day

7day 28day

18day

14day

14day

21day

21day(a)

(b)

(c)












1(phenyl phenoxy)







Acknowledgment


References
































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















Glycine

Methyl 35 days [37]

Ethyl




Alanine

Methyl 5 weeks [37]











Com

pres

sive s

treng

th (M

Pa)

PNEA PNEA50 mPh50

lowast


100

80

60

40

20

0


50

mPh50


80

PhPh20


50

PhPh50










14(imidazolyl)

06phosp-






1(p-









122(GlyGlyOH)

007

(GlyGlyODOX)005

(AMPE G550)066


50)





1hr

4hr

1hr

1hr 3hr 6hr

7day

7day 28day

18day

14day

14day

21day

21day(a)

(b)

(c)












1(phenyl phenoxy)







Acknowledgment


References
































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















Com

pres

sive s

treng

th (M

Pa)

PNEA PNEA50 mPh50

lowast


100

80

60

40

20

0


50

mPh50


80

PhPh20


50

PhPh50










14(imidazolyl)

06phosp-






1(p-









122(GlyGlyOH)

007

(GlyGlyODOX)005

(AMPE G550)066


50)





1hr

4hr

1hr

1hr 3hr 6hr

7day

7day 28day

18day

14day

14day

21day

21day(a)

(b)

(c)












1(phenyl phenoxy)







Acknowledgment


References
































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















14(imidazolyl)

06phosp-






1(p-









122(GlyGlyOH)

007

(GlyGlyODOX)005

(AMPE G550)066


50)





1hr

4hr

1hr

1hr 3hr 6hr

7day

7day 28day

18day

14day

14day

21day

21day(a)

(b)

(c)












1(phenyl phenoxy)







Acknowledgment


References
































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















1(p-









122(GlyGlyOH)

007

(GlyGlyODOX)005

(AMPE G550)066


50)





1hr

4hr

1hr

1hr 3hr 6hr

7day

7day 28day

18day

14day

14day

21day

21day(a)

(b)

(c)












1(phenyl phenoxy)







Acknowledgment


References
































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















1hr

4hr

1hr

1hr 3hr 6hr

7day

7day 28day

18day

14day

14day

21day

21day(a)

(b)

(c)












1(phenyl phenoxy)







Acknowledgment


References
































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















1(phenyl phenoxy)







Acknowledgment


References
































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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Review Article Biodegradable Polyphosphazene Biomaterials...

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