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Review
Advanced drug and gene delivery systems based on functionalbiodegradable polycarbonates and copolymers
Wei Chen a,b, Fenghua Meng a, Ru Cheng a, Chao Deng a, Jan Feijen a,b,⁎, Zhiyuan Zhong a,⁎⁎a Biomedical Polymers Laboratory, Jiangsu Key Laboratory of Advanced Functional Polymer Design and Application, College of Chemistry, Chemical Engineering and Materials Science,Soochow University, Suzhou 215123, PR Chinab Department of Polymer Chemistry and Biomaterials, Faculty of Science and Technology, MIRA Institute for Biomedical Technology and Technical Medicine, University of Twente, P.O. Box 217,7500 AE Enschede, The Netherlands
Article history:Received 4 February 2014Accepted 13 May 2014Available online 21 May 2014
Biodegradable polymeric nanocarriers are one of the most promising systems for targeted and controlled drugand gene delivery. They have shown several unique advantages such as excellent biocompatibility, prolonged cir-culation time, passive tumor targeting via the enhanced permeability and retention (EPR) effect, and degradationin vivo into nontoxic products after completing their tasks. The current biodegradable drug and gene delivery sys-tems exhibit, however, typically low in vivo therapeutic efficacy, due to issues of low loading capacity, inadequate
in vivo stability, premature cargo release, poor uptake by target cells, and slow release of therapeutics insidetumor cells. To overcome these problems, a variety of advanced drug and gene delivery systems has recentlybeen designed and developed based on functional biodegradable polycarbonates and copolymers. Notably,polycarbonates and copolymers with diverse functionalities such as hydroxyl, carboxyl, amine, alkene, alkyne,halogen, azido, acryloyl, vinyl sulfone, pyridyldisulfide, and saccharide, could be readily obtained by controlledring-opening polymerization. In this paper, we give an overview on design concepts and recent developmentsof functional polycarbonate-based nanocarriers including stimuli-sensitive, photo-crosslinkable, or activetargeting polymeric micelles, polymersomes and polyplexes for enhanced drug and gene delivery in vitro andin vivo. These multifunctional biodegradable nanosystems might be eventually developed for safe and efficientcancer chemotherapy and gene therapy.
In the past decade, ever-growing efforts have been devoted to thedevelopment of drug delivery nanotechnology since it offers a suitableapproach for transporting low molecular weight drugs includingchemotherapeutics and peptides, as well as macromolecules such asproteins or genes by either localized or targeted delivery to the diseasedtissue [1–3]. Drug delivery nanotechnology focuses on formulating ther-apeutic agents in biocompatible and biodegradable nanocarriers such aspolymeric micelles, nanoparticles, nanocapsules, polymersomes andpolymer conjugates. These nanosized polymeric systems have demon-strated multifaceted advantages in drug delivery, including (i) aremarkable enhancement of the aqueous solubility of poorly solubleanti-cancer drugs; (ii) prolonging drug circulation time and preventingdrug degradation, especially for therapeutic agents like proteins,
d gene delivery nanosystems based
peptides, and nucleic acid drugs; (iii) passive targeting to tumor tissuesvia the enhanced permeability and retention (EPR) effect [4–6]. In addi-tion, triggered release of therapeutic agents from nanoparticles can beinduced by modulating polymer characteristics, allowing the desiredtherapeutic efficacy in the target tissue [7–11]. Furthermore, enhanceddrug delivery to the target sites can be accomplished via conjugationof nanoparticles with a biospecific ligand [12–14].
Among numerous biodegradable polymeric materials used in drugdelivery applications, aliphatic polycarbonates are one of the most in-teresting materials due to their excellent biocompatibility, nontoxicdegradation products, and absence of autocatalytic degradation pro-cesses [15–17]. For example, based on their favorable material proper-ties, copolymers of cyclic carbonates such as trimethylene carbonate(TMC) with cyclic ester monomers such as lactide (LA), glycolide (GA),and ε-caprolactone (ε-CL) have already found application as sutures and
on functional biodegradable polycarbonates and copolymers.
O
O
OO O O
O
Om n
O
OO
OO O O
O
O O
m n
RR
R
O
OH
OO
O O
O
m
Pd/C/H2
PEG-b-PBTMC
O
O
OH OH
p q n
PEG-b-P(T)MBPEC
RR
R
O O
O O
O O
O O
O O
O
O
O
O
O
O
OO
OH
OH
O
OH
O
O
HEHDO-star-PEG
HEHDO
PEG-b-P(LA-co-DHP)
(1)
(2)
(3)
PEG-OH
PEG-OH
PEG-OH LA
PEG-NCOSCROP
120 oC
BTMC
TMBPEC: R = OCH3BPEC: R = H
(R = H)
EHDO
(R = OCH3)
Scheme 2. Synthesis of hydroxyl-functionalized polycarbonates and copolymers.
400 W. Chen et al. / Journal of Controlled Release 190 (2014) 398–414
in controlled drug delivery systems [18–21]. Notably, polycarbonates aredegraded in vivo by surface erosion in contrast to the bulk degradation be-havior observed with aliphatic polyesters [22]. Furthermore, polycarbon-ate degradation will not lead to increased levels of acidity, which mayoccur during polyester degradation, andwhichmaybehazardous to load-ed drugs or healthy tissues.
However, in the practice of drug delivery, very often PTMC polymersand their copolymers cannot satisfy the requirements for particularapplications, due to their high hydrophobicity, improper degradationprofile, and/or lack of reactive centers in the polymer chain for the cova-lent immobilization of bioactive molecules such as drugs, peptides andproteins. In the past decade, the design of functional cyclic carbonatemonomers has receivedmore andmore interest, and various functionalaliphatic polycarbonate-based polymers and copolymers containing e.g.hydroxyl, carboxyl, and amine pendant groups have been reported[23–26]. These functional polymers on the one hand show improvedphysicochemical properties such as enhanced hydrophilicity and
Table 1Functional nanocarriers based on hydroxyl-containing polycarbonates and copolymers.
Polymers Nanosystems M
Type Size (nm)
PEG-b-PBTMC Micelle 96 EPEG5k-b-PTMBPEC3.7k Micelle 120 DPEG1.9k-b-PTMBPEC6.0k Polymersome 120 D
PPEG-SS-PTMBPEC Micelle 140 DPEG-b-P(LLA-co-DHP) Prodrug 74 DHEHDO-star-PEG Micelle 120 D
biodegradability, and on the other hand facilitate drug conjugation orfurther derivatization. Herein, we review up-to-date novel functionalpolycarbonate-based biodegradable nanocarriers for enhanced drugand gene delivery (Scheme 1). It is anticipated that with the favorableproperties of biodegradable polycarbonates and copolymers we areable to create advanced multifunctional drug carriers for targeted, safeand efficient cancer treatment.
2. Functional nanocarriers based on hydroxyl-containingpolycarbonates and copolymers
Hydroxyl-containing polycarbonates and copolymers are usuallyprepared by homopolymerization of benzyloxy [27–30] or acetal[31–35] protected cyclic carbonates or copolymerization of these func-tional cyclic carbonates with other cyclic monomers (e.g. LA, ε-CL, andTMC), followed by deprotection with Pd/C or hydrolysis at mild acidicconditions (Scheme 2). Recently, biodegradable hyperbranched
odel drugs Special advantages Refs.
llipticine High stability and drug loading ability [41,42]OX PTX pH-sensitivity [44]OX∙HCl/TX
pH-sensitivity [45]
OX pH and reduction dual-sensitivity [46]OX pH-sensitivity and tumor targetability [50]OX High stability and drug loading ability [52]
Fig. 1. pH-sensitive degradable polymersomes based on PEG-b-PTMBPEC block copolymer for triggered release of both hydrophilic and hydrophobic anticancer drugs. In com-parison, pH-sensitive degradablemicelles/nanoparticles are typically applied for release of hydrophobic drugs only. [45], Copyright 2010. Reproduced with permission from Elsevier Ltd.
401W. Chen et al. / Journal of Controlled Release 190 (2014) 398–414
polycarbonates were synthesized by ring-opening polymerization ofhydroxyl-bearing cyclic carbonate, using its own hydroxyl group asthe initiator, to produce hydroxyl functional groups at the outer sphereof the hyperbranched polymer [36,37]. These hydroxyl-enrichedpolycarbonates demonstrated high cell-biocompatibility using COS 7cells as shown by the MTT assay [38]. The degradation rate of aliphaticpolycarbonates is considerably lower than that ofmost aliphatic polyes-ters, which restricts their use as short-term implant biomaterials. Due tothe improved hydrophilicity and the autocatalytic effect of the hydroxylgroups, hydroxyl-enriched polycarbonates have amuch faster degrada-tion rate than that of the non-functionalized PTMC analog, with a simi-lar structure differing merely by the absence of pendant hydroxylgroups [32,38,39]. It is assumed that the fast degradation also involvesan intra-molecular nucleophilic attack by the pendant hydroxyl groups
Fig. 2. Schematic representation of multifunctional copolymer micelles targeting lactose- orpermission from Elsevier Ltd.
on the carbonate linkages of the main chain [40]. These hydroxyl-containing functional polycarbonates and copolymers can be facilelyconstructed to advanced nanocarriers for drug delivery (Table 1).
Well-defined amphiphilic biodegradable block copolymers compris-ing of a functionalized polycarbonate hydrophobe and poly(ethyleneglycol) (PEG) undergo phase separation in aqueous media, leading tothe formation of nanosized core–shell micellar structures. For example,Allen et al. reported amphiphilic diblock copolymers with various blockcompositions of PEG as the hydrophilic segment and poly(5-benzyloxytrimethylene carbonate) (PBTMC) as the hydrophobic segment [41],in which PEG-b-poly(5-benzyloxy trimethylene carbonate) (PEG-b-PBTMC) copolymers self-assembled into micelles in water with a nar-row size distribution. Ellipticine-loaded PEG-b-PBTMC (5 k-b-4.8 k) mi-celles had an average diameter of 96 nmand a drug loading efficiency of
galactose-receptors on mammalian liver cells. [51], Copyright 2010. Reproduced with
O O
O
OO O O
O
m n
PEG-b-PBTMCC
O
O
PEG-OH
O
O
Pd/C/H2 OO O O
O
m n
PEG-b-PTMCCOH
O
BTMCC
PEG-OH,LA Pd/C/H2
PEG-b-P(TMCC-co-LA)
OO O O
O
m p
OH
O
O
Oq n
O O
O
m
PTMCCOH
O
ROP Pd/C/H2 PEIO O
O
m
PTMCC-g-PEI
NH
O
PEI
Conjugation
Scheme 3. Synthesis of carboxyl-functionalized polycarbonates and copolymers.
402 W. Chen et al. / Journal of Controlled Release 190 (2014) 398–414
95% (Table 1). In vitro release studies showed that approximately 32% ofellipticine was released in 24 h in phosphate-buffered saline (PBS,10mM, pH7.4) at 37 °C, while 60% of drugwas released in the presenceof serum protein under otherwise the same conditions [42].
Based on acid-labile acetal or ketal groups, pH-sensitivemicellar sys-tems have been developed for controlled drug release [43]. For instance,we designed a novel acid-labile acetal-containing cyclic carbonatemonomer, 2,4,6-trimethoxybenzylidenepentaerythritol carbonate(TMBPEC) to obtain pH-sensitive degradable PEG-b-PTMBPEC blockcopolymer micelles [44]. The results showed that the acetals in themicelles were prone to fast hydrolysis at mildly acidic pH, whichtransformed the hydrophobic PTMBPEC block into a hydrophilicpoly(pentaerythritol carbonate) (PPEC) block, resulting in markedlyenhanced drug release. Interestingly, pH-sensitive degradablepolymersomes were prepared when PEG-b-PTMBPEC block copolymerhad a molecular weight of 1.9 k-b-6.0 k (Fig. 1) [45]. Both paclitaxel(PTX, hydrophobic) and doxorubicin hydrochloride (DOX HCl, hydro-philic) could simultaneously be loaded into polymersomes (Table 1).In vitro release studies demonstrated that both PTX and DOX HCl werereleased in a pH-dependent manner. Very recently, we also developedredox and pH-responsive degradable nanoparticles based on PEG-SS-PTMBPEC block copolymers for dually triggered intracellular release ofdoxorubicin (DOX) [46]. The in vitro release studies showed that94.2% of DOX was released from the dual-responsive biodegradablenanoparticles in 10 h in the presence of 10 mM glutathione (GSH) atpH 5.0. Interestingly, DOX release was obviously enhanced by 2 or 4 hincubation at pH 5.0 and then at pH 7.4 with 10 mM GSH (mimickingthe intracellular pathway of endocytosed micellar drugs). MTT assaysusing HeLa and RAW 264.7 cells revealed that DOX-loaded PEG-SS-PTMBPEC nanoparticles had a significant anti-tumor activity.
Table 2Functional nanocarriers based on carboxyl-containing polycarbonates and copolymers.
The benzyloxy group is inert during ring-opening polymerization(ROP) and is therefore often used to protect hydroxyl functional groups.After deprotection, hydroxyl-functionalized aliphatic polycarbonatescan be obtained and used for the covalent immobilization of bioactivemolecules such as drugs, peptides and proteins [47–49]. For example,Jing et al. prepared DOX prodrugs from amphiphilic PEG-b-poly(L-lactide-co-2,2-dihydroxymethyl propylene carbonate) [PEG-b-P(LLA-co-DHP)] copolymer using either a carbamate or acid-labile hydrazonelink. The tumor-targeting ligand folic acid (FA) was also conjugated toPEG-b-P(LLA-co-DHP) via its hydroxyl group [50]. The cellular uptakestudies showed an enhanced internalization of FA-containing micellesby the human ovarian cancer cell line SKOV-3 as compared to thecontrol without FA. In a following study, they have also conjugatedlactose and Rhodamine B (RhB) to PEG-b-P(LLA-co-DHP) [51]. Theex vivo imaging studies showed that thus formedmicelles preferentiallyaccumulated in the liver of mice (Fig. 2).
Feng et al. prepared amulti-arm hyperbranched copolymer (HEHDO-star-PEG) by conjugating PEG to hydroxyl-enriched hyperbranched ali-phatic polycarbonates based on 5-ethyl-5-hydroxymethyl-1, 3-dioxan-2-one (EHDO) via urethane links [52]. HEHDO-star-PEGmicelles showedexcellent stability, good cell-compatibility, and high loading capability ofDOX, and a sustained DOX release pattern.
3. Functional nanocarriers based on carboxyl-containingpolycarbonates and copolymers
Carboxyl-containing polycarbonates and copolymers are usuallyprepared by ROP of carboxyl-protected cyclic carbonate monomer,followed by deprotection using Pd/C. Benzyl ester groups are oftenused to protect carboxyl functional groups [53–55] (Scheme 3).
Special advantages Refs.
High stability and drug loading ability [56]YA/GEF rapamycin Improved drug loading ability [57–60]
Targeting immuno-therapy and chemotherapy [63–65]-Pt pH-sensitivity and tumor targetability [66–68,73]
Fig. 3. The preparation of DOX-conjugated immuno-nanoparticles. [64], Copyright 2009. Reproduced with permission fromWiley-VCH Verlag GmbH & Co. KGaA.
403W. Chen et al. / Journal of Controlled Release 190 (2014) 398–414
Inadequate in vivo stability is a practical issue for micellar drugdelivery systems [16]. Mahato and coworkers reported that PEG-b-poly(2-methyl-2-carboxy trimethylene carbonate-g-dodecanol) (PEG-b-PCD) micelles with a strong hydrophobic inter-chain interactionwithin the micellar core had a remarkably high stability with a lowcritical micelle concentration (CMC) of 10−8 M, which was 50-foldlower than that of the PEG-b-poly(2-methyl-2-carboxy trimethylenecarbonate) (PEG-b-PTMCC) counterpart [56]. Micelles of PEG-b-poly(2-methyl-2-benzoxycarbonyl-propylene carbonate-co-L-lactide)copolymer [PEG-b-P(BTMCC-co-LLA)] containing 20 mol% carbonateunits could markedly increase the aqueous solubility of bicalutamidefrom 5 mg/mL to 4000 mg/mL and exhibited about 4-fold higherloading of bicalutamide than PEG-b-PLLA micelles [57] (Table 2). In afollowing study, PEG-b-P(BTMCC-co-LLA) micelles were applied fordelivery of (2-(1 H-Indol-5-yl) thiazol-4-yl) 3,4,5-trimethoxyphenylmethanone (LY293) for the treatment of resistant melanoma [58]. Theresults showed that LY293-loaded micelles exhibited low IC50 valuesof 12.5 nM and 25 nM against A375 and B16F10 cells, respectively(Table 2). PEG-b-P(BTMCC-co-LLA)micelles co-loadedwith cyclopamine(CYA, hedgehog (Hh) inhibitor) and gefitinib (GEF, epidermal growthfactor receptor (EGFR) inhibitor) showed a synergistic effect againstL3.6pl cells while an additive effect against MIA PaCa-2cells [59]. Thein vivo studies revealed that Hh and GEF-loaded micelles decreasedtumor growth rate in mice with L3.6pl-derived xenografts. PEG-b-PBTMCC micelles have also been investigated for rapamycin delivery[60].
Shoichet and coworkers prepared poly(2-methyl-2-carboxytrimethylene carbonate-co-D,L-lactide) [P(TMCC-co-DLLA)] copoly-mer nanoparticles and investigated their serum stability [61,62].Immuno-polymeric micelles were obtained by grafting amine-PEG-furan (NH2-PEG-furan) to P(TMCC-co-DLLA) [P(TMCC-co-DLLA)-g-PEG-
Table 3Functional nanocarriers based on amino, amine, or urea-containing polycarbonates and copoly
furan] followed by conjugating maleimide-modified anti-HER2 (a thera-peutic antibody used to treat breast cancer) to the furan groups at thesurface of micelles by Diels–Alder cycloaddition [63]. These immuno-micelles were shown to specifically bind to HER2-over-expressing cells.In a following study, DOX-conjugated immuno-micelles were preparedby also attaching DOX-maleimide conjugate to the surface of micelles[64]. Flow cytometric analysis showed that they induced similar apopto-tic activity in SKBR-3 cells as free DOX (Fig. 3). It should be noted thatDOX-anti-HER2 nanoparticle was significantly more cytotoxic againstSKBR-3 cells than against healthy HMEC-1 cells. Moreover, DOX-conjugated immuno-micelles were obviously more efficacious thanDOX or anti-HER2-conjugated micelles. Similarly, RGD-functionalizedmicelles that target to corneal epithelial cells through RGD-cell surfacereceptors were prepared by treating P(TMCC-co-DLLA)-g-PEG-N3
micelles with alkyne-bearing KGRGDS peptides [65].Jing and coworkers conjugated docetaxel and RGD to PEG-b-poly(2-
methyl-2-carboxy trimethylene carbonate-co-L-lactide) [PEG-b-(PTMCC-co-LLA)] copolymers through their functional carboxyl groups [66,67].The resulting nanoparticles showed high cytotoxic activity against HeLacancer cells. Using their carboxyl functional groups, pirarubicin (THP)was successfully conjugated onto PEG-b-(PTMCC-co-LLA) copolymer viahydrazone, ester, and amide bonds, respectively [68]. The in vitro releasestudies showed that THP-conjugated micelles through a hydrazone bonddisplayed the highest pH sensitivity, inwhich 40% THPwas released at pH5.0 in 40 h (Table 2). The in vivo experiments in Balb/cmicemodels bear-ing EMT6 tumors demonstrated that THP-conjugated micelles through ahydrazone bond had the highest anti-tumor activity while those withan amide linkage had the lowest activity. The conjugation of dopamineto the carboxyl groups in PEG-b-poly(2-methyl-2-carboxy trimethylenecarbonate)-b-poly(L-lactide) (PEG-b-PTMCC-b-PLLA) gave PEG-b-P(TMCC-g-dopamine)-b-PLLA copolymer that could be self-assembled
mers.
del drugs Special advantages Refs.
profen pH-sensitivity [83]profen pH-sensitivity [84]dnisone acetate pH-sensitivity [86]T, DOX pH-sensitivity [87]le red pH-sensitivity [88]X thioridazine High stability and drug loading ability [91–97]
(1)
PEG-b-P(CA-co-LA)
OO O O
O
m p
O
Oq n
O O
O
HN
O
O
Bn
CAB
PEG-OHLA HBr/HAc
NH2
O O
O
N
(2)
DMATC
ROPO O
O
mN
PDMATC
(3)
OO
O
N
N
O
OO
CLO O
N
O
O
Op q n
ADMC
P(ADMC-co-CL)
TMCC/TMC
P(TMC-co-ADMC-co-TMCC)
O O ON
O
Op q n
O O
O OrO
HO
PEG/CLO O
N
O
O
Op q n
OO m
PEG-b-P(ADMC-co-CL)
EHDO
SCROP
O
OO
H(EHDO-co-ADMC)
O O
O
O
O
O O
OH
O
O
O
O
O
O
N O
O
O
O
ON
O O
O
O
O
O O
O
UMTC
(4)
O
O
NH
NHPh
O
PEG-OH OO O O
O
m nO
O
NH
NHPh
O
PEG-b-PUMTC
PEG-OHBTMCC Pd/C/H2
PEG-b-P(TMCC-co-UMTC)
OO O O
O
m p
OH
O
O q nO
O
O
NH
NHPh
O
O
Scheme4. Synthesis of amino, amine, orurea-functionalizedpolycarbonates and copolymers.
Fig. 4. Biodegradable supramolecular nanostructures based on acid/urea-functionalizedmixedmReproduced with permission from Elsevier Ltd.
404 W. Chen et al. / Journal of Controlled Release 190 (2014) 398–414
into micelles and readily crosslinked by bubbling air due to oxidation ofthe dopamine groups [69]. Zhuo and coworkers prepared phenylboronicacid (BA)-functionalized amphiphilic block copolymer, Pluronic-b-poly(2-methyl-2-carboxy trimethylene carbonate-g-phenylboronic acid)(Pluronic-b-PTMCC-g-BA) by conjugating 3-aminophenylboronic acid tothe carboxylic acid groups in Pluronic-b-PTMCC [70]. The results showedthat BA groups located at themicelle surface could recognize HepG2 cellsand promote the intracellular uptake of DOX-loaded micelles.
Platinum(II)-baseddrugs such as cisplatin, carboplatin, andoxaliplatinare widely employed for the treatment of various cancers including ovar-ian, lung and testicular cancers [71,72]. Jing et al. reported that PEG-b-P(TMCC-co-LLA) copolymers could actively load diaminocyclohexaneplatinum (DACH-Pt) to form PEG-b-P(LLA-co-TMCC/Pt) complexes [73].FA-functionalized micelles could be prepared by co-assembly of PEG-b-P(LLA-co-TMCC/Pt) and FA-PEG-b-PLLA copolymers. The in vivo studiesin mice showed that DACH-Pt-micelles had a longer blood circulationtime than free oxaliplatin and FA-decorated DACH-Pt-micelles exhibitedgreater antitumor efficacy than non-targeting DACH-Pt-micelles or freeoxaliplatin.
Functional polycarbonates are of great interests for gene delivery. Forexample, Yang and coworkers developed biodegradable cationicpolycarbonates by conjugating various oligoethyleneimines (OEIs) suchas triethylenetetramine, tetraethylenepentamine or pentaethylene-hexamine onto carboxylic acid-functionalized polycarbonates viacarbodiimide chemistry [74]. In-vitro gene transfection studies demon-strated that OEI-functionalized polycarbonates could mediate efficientluciferase expression in HEK293, HepG2 and 4T1 cell lines at levelsthat were comparable, or even superior, to that of the polyethylenimine(PEI) standard (Table 2). In a similar way, Chen et al. conjugated OEIsonto PEG-b-PTMCC amphiphilic copolymer via EDC/NHS chemistry[75]. The introduction of PEG could improve solubility, reduce aggrega-tion, decrease cytotoxicity, and possibly decrease opsonization byserum proteins in the bloodstream. In vitro experiments showed thatPEG-b-PTMCC-OEI1800 conjugate exhibited lower cytotoxicity andhigher gene transfection than PEI-25K in CHO and COS-7 cell lines inthe absence as well as in the presence of serum.
icelles designed for high cargo loading capacity and kinetic stability. [93], Copyright 2010.
(1)
PEG-b-P(LA-co-MAC)
PEG O O O
O
LAp q n
O O
O
MAC
PEG-OHLA
R
O
O
OO
AIBNCrosslinking
"thiol-ene"reaction
(R = CH3)
PEG O O O
O
LAp q nO
OR1-SH
PEG-b-P(LA-co-MACR1)
SR1
(R = H)
DTC
P(MAC-co-DTC)
O O
O
DTCp q nO
O
EpoxidizationO O
O
DTCp q nOO
O
P(MACO-co-DTC)
O O
O
AC
OO
PEG-OH
CL
LA orCL O
O O O
O
PLA (or PCL)m n
O
O
UVirradiation
Crosslinking
PEG-b-PAC-b-PLA or PEG-b-PAC-b-PCL
PEG-OHTMC
OO O O
O
m p
O
O q nO
PEG-b-P(TMC-co-AC)O
O
PEG-OHTMBPEC O
O O O
O
m p
O
O q nO
PEG-b-P(TMBPEC-co-AC)O
O
O O
Ph(OMe)3
UVirradiation
Crosslinking
Michae-typeaddition
PEG-b-P(TMC-co-ACR2)R2-SH
O p
O
O q nO
P(CL-co-AC)O
O
O
1. Michae-typeaddition
PCL-g-PHEMA2. CPADNconjugation
HEMA
RAFTpolymerization
(2)
Scheme 5. Synthesis of allyl or acryloyl-functionalized polycarbonates and copolymers.
405W. Chen et al. / Journal of Controlled Release 190 (2014) 398–414
4. Functional nanocarriers based on amino, amine, orurea-containing polycarbonates and copolymers
In the past years, cyclic carbonates with pendant amide, amino,amido and urea functionalities have been reported [76–80] (Table 3).Cyclic carbonates with pendant amides include those with pendantcarbamic acid benzyl ester (Z) groups and carbamic acid tert-butylester (Boc) functional groups (Scheme 4). For example, Jing et al. pre-pared degradable poly(ester-co-carbonate)s with benzyloxycarbonylprotected amino groups, which following catalytic hydrogenationafforded poly(ester-co-carbonate)s with free amino groups [78]. RGDcould be readily conjugated to amino-functionalized poly(ester-co-carbonate)s via carbodiimide chemistry. FA and fluorescent probe-functionalized amphiphilic degradable copolymers could be preparedfrom amino-functionalized PEG-b-poly(L-lactide-co-serinol carbonate)
Table 4Functional nanocarriers based on alkene or alkyne-containing polycarbonates and copolymers
[PEG-b-P(LLA-co-CA)] using NHS-activated FA and fluorescein isothio-cyanate (FITC), respectively [81]. Feng et al. prepared water-solublepoly(2-dimethylaminotrimethylene carbonate) (PDMATC) by ROP of anew cyclic carbonate monomer, 2-dimethylaminotrimethylene carbon-ate (DMATC), using Novozym-435 as a catalyst [82].
Feng and coworkers designed a novel cyclic carbonate monomer,6,14-dimethyl-1,3,9,11-tetraoxa-6,14-diaza-cyclohexadecane-2,10-dione [(ADMC)2], and synthesized the corresponding polycarbonate,poly(6,14-dimethyl-1,3,9,11-tetraoxa-6,14-diaza-cyclohexadecane-2,10-dione) (PADMC), viaNovozym-435 lipase or tin octoate [Sn(Oct)2]catalyzed ring-opening polymerization [79]. The copolymerization of(ADMC)2 and ε-CL provided amphiphilic degradable P(ADMC-co-CL)copolymers containing tertiary amine groups in the backbone [83].P(ADMC-co-CL) copolymers with a higher ADMC content degradedfaster in PBS solution (pH 7.4, 100 mM) at 37 °C. P(ADMC-co-CL)-based microspheres exhibited a controlled release of ibuprofen, whichis accelerated at acidic conditions (Table 3). In a following study,PEG was introduced into P(ADMC-co-CL) copolymer [84]. The resultingcopolymers could readily self-assemble into micelles (~100 nm).Ibuprofen-loaded PEG-b-poly(ADMC-co-CL) micelles showed an accel-erated drug release at acidic conditions due to the protonation of tertia-ry amine groups in ADMC units. In a similar way, amphoteric aliphaticpolycarbonate bearing both amine and carboxyl groups was synthe-sized by enzymatic copolymerization of TMCC, (ADMC)2 and TMC[85]. The simultaneous introduction of amine and carboxyl functionali-ties provided copolymers with pH-tunable self-aggregation, leading towell-dispersed positively or negatively charged nanoparticles in acontrolled manner. Taking advantage of strong buffering capacity andfast degradation of PADMC, amphiphilic PCL-b-PADMC-b-PCL triblockcopolymer were prepared to form micelles for pH-sensitive releaseof prednisone acetate [86]. Very recently, nanoparticles based onamine-functionalized poly(6,14-dimethyl-1,3,9,11-tetraoxa-6,14-diaza-cyclohexadecane-2,10-dione)-b-poly(1,3-dioxepan-2-one) (PADMC-b-PTeMC) block copolymer were found to be efficiently taken up by cellsto give accelerated intracellular DOX release in HeLa cells at lower pH[87].
Based on hyperbranched hydroxyl-enriched aliphatic polycarbonate[36], Feng et al. constructed pH-sensitive macro and nano-gels from hy-droxyl and amine-functionalized hyperbranched P(EHDO-co-ADMC)[88]. These nanogels could stabilize poorly water-soluble moleculessuch as nile red in a neutral pH environment. Notably, a fast change inthe size and morphology of the nanogels took place across a narrowpH range from 7.4 to 6.6 due to the ionization of the tertiary aminegroups in the ADMC units.
Urea groups can bind carbonyl derivatives such as DOX and PTX, andtheir isosteres (sulfonates, phosphonates, phosphates, etc.) [89,90].Therefore,micelles containingurea groups often showahigh loading ef-ficiency for carbonyl-containing anticancer drugs. Yang and coworkers
.
Model drugs Special advantages Refs.
– Thermal-crosslinking [111]PTX UV-crosslinking and tumor targetability [108]PTX UV-crosslinking and tumor targetability [109]PTX UV-crosslinking and pH-sensitivity [112]DOX Hydrogen bonding formation [113]DOX Hydrogen bonding crosslinking [114]DOX pH-sensitivity [115]DOX Combination of ROP and RAFT [116]Protein High protein loading and pH-sensitivity [117]Protein Reduction-sensitivity [118]Gene Gene delivery [119,120]DOX pH-sensitivity [121]
406 W. Chen et al. / Journal of Controlled Release 190 (2014) 398–414
prepared urea-functionalized amphiphilic block copolymers fromorganocatalytic ring-opening copolymerization of urea-functionalizedcyclic carbonates (UMTC) and ethoxycarbonyl cyclic carbonate(EMTC) using PEG as a macroinitiator [91,92]. The hydrogen-bondingfunction of the urea groups considerably improved the kinetic stabilityof both drug-free and DOX-loaded micelles. The higher urea contentsalso led to a significantly enhanced DOX loading and a slightly de-creased drug release rate. To further fine tune the critical properties ofdrug-loaded micelles, they prepared DOX-loaded mixed polymeric mi-celles of PEG-b-PTMCC/PEG-b-PUMTC copolymers [93]. The mixed mi-celles demonstrated higher kinetic stability than micelles preparedfrom a single polymer, due to hydrogen bonding between the carboxyl-ate and urea groups in the polycarbonate (Fig. 4). They also found thatthe molecular structure of acid- and urea-functionalized polycarbonateblock copolymers has an effect on the micellar properties, such as CMC,particle size and size distribution, kinetic stability and drug loading ca-pacity [94]. The polymers with carboxyl and urea groups placed inthe random form formed micelles with better size distribution(two size populations), and their DOX-loaded micelles were morestable. In addition, the intravenous injection of DOX-loaded micellesinto mice yielded lower DOX accumulation in the heart tissue thanfree DOX formulation. Recently, they investigated co-delivery of thi-oridazine (THZ) and DOX for the treatment of both cancer cells andcancer stem cells using mixed acid- and urea-functionalized micelles[95]. The co-delivery of DOX-loaded mixed micelles (DOX-MM) andTHZ-loaded mixed micelles (THZ-MM) produced a stronger inhibi-tory effect on sorted BT-474 and MCF-7 human breast cancer stemcells as compared to single DOX-formulation. The in vivo studiesusing a BT-474 xenografted female BALB/c nude mouse model dem-onstrated that the co-delivery of DOX-MM and THZ-MM inhibitedthe tumor most efficiently and presented the strongest activityagainst cancer stem cells.
Fig. 5. Illustration of photo-crosslinked pH-sensitive degradable micelles based on PEG-b-P(TMexhibit superior extracellular stability andminimal drug leakage on dilution while “actively” relments. [112], Copyright 2012. Reproduced with permission from Elsevier Ltd.
The kinetic stability of micelles is an important factor that influencesmicelle behavior in the blood stream as it determines how fast themicelles will dissociate into individual polymer molecules [96]. Themicelles may be still stable in the blood stream for a certain period oftime even at polymer concentrations below its CMC. Yang and co-workers investigated the effect of kinetic stability on biodistributionand antitumor activity of drug-loaded biodegradable polymericmicelles[97]. DOX-loaded PEG5Kmixedmicelles had higher kinetic stability thanDOX-loaded PEG10K mixedmicelles due to the higher hydrophobicity ofthe PEG5K block copolymers. The in vivo studies conducted with a 4T1mouse breast cancer model demonstrated that the PEG5k mixed mi-celles were preferably transported to the tumor, accompanied with afaster accumulation in the tumor and to a larger extent than thePEG10k mixed micelles. DOX-loaded PEG5k mixed micelles inhibitedtumor growth more effectively than the corresponding DOX-loadedPEG10k mixed micelles without causing significant body weight loss orcardiotoxicity.
5. Functional nanocarriers based on alkene or alkyne-containingpolycarbonates and copolymers
In the past few years, synthesis of functional polycarbonates con-taining pendant unsaturated alkene and alkyne groups has receivedincreasing attention (Scheme 5). Here, no protection/deprotectionsteps are needed. Moreover, alkene and alkyne groups allow furtherpost-polymerization modification via highly efficient and orthogonal re-actions, such as Michael addition [98–101], radical thylation [102–104],epoxidation [105,106], and thermal or UV-crosslinking [107–110].
Micelle crosslinking can efficiently inhibit micelle disassociationand premature drug release after intravenous injection. Jing et al. pre-pared crosslinked biodegradable micelles via self-assembly of PEG-b-poly(L-lactide-co-5-methyl-5-allyloxycarbonyl-1,3-dioxan-2-one) block
BPEC-co-AC) block copolymer. PTX-loaded crosslinked pH-sensitive degradable micelleseasing PTX undermildly acidic conditionsmimicking that of the endo/lysosomal compart-
(1)
O O
O
BrBr
CLO p
O
O q nO
P(DBTC-co-CL)
OBr Br
NaN3 PEG-alkynl
Click chemistryO p
O
O q nO
P(DTC-co-CL)-g-PEG
ON NN
N
OPEG
O NN
O
PEG
O
DBTC
O O
O
N3N3
ADTC
OO O O
O
m p
O
O q nO
PEG-b-P(DTC-co-ADTC)
N3 N3Click chemistry
O
O
14PEG-b-P(DTC-ADTC-g-Pal)
PEG-OH/DTC
ROPO
O nO
N3 N3
CuBrMicrowave, 70 oC
R
O
O nO
N NN
N NN
R R
CH2OHR: (CH2)3COOH
CH2NH2
PADTC
(2)
O O
O
O
O
X
MTC-X
ROPO
O nOO
OX
NN O
O nOO
ON+ NX-
P(MTC-X) P(MTC-N+X-)(X = Cl, Br, I)
(3)
PADTC-g-R
Scheme 6. Synthesis of halogen or azido-functionalized polycarbonates and copolymers.
407W. Chen et al. / Journal of Controlled Release 190 (2014) 398–414
copolymer [PEG-b-P(LLA-co-MAC)] followed by radical polymerizationof allyl groups with AIBN [111]. The crosslinked micelles had a narrowsize distribution and similar sizes to their parent uncrosslinked micelles.We prepared interfacially crosslinkedmicelles from PEG-b-poly(acryloylcarbonate)-b-poly(D,L-lactide) (PEG-b-PAC-b-PDLLA) and PEG-b-poly(acryloyl carbonate)-b-polycaprolactone (PEG-b-PAC-b-PCL), re-spectively [108,109]. This interfacial crosslinking approach has uniquelycombined advantages of core and shell crosslinking, which on the onehand allows the crosslinking reaction taking place at high micelle con-centrations without inter-micellar crosslinking, and on the other handhas little influence on the properties of the micellar core and shell. Theinterfacially crosslinked micelles exhibited excellent stability with mini-mal drug release at low micellar concentrations (Table 4). Notably,in vivo studies using human hepatoma-bearing nude mice revealedthat galactose-decorated PTX-loaded crosslinked PEG-b-PAC-b-PCL mi-celles inhibited the growth of the human hepatoma more effectivelythan PTX-loaded crosslinked micelles without galactose decorationas well as galactose-decorated PTX-loaded non-crosslinked micelles[109], confirming that micelle stabilization plays a critical role intargeted tumor therapy. In a more recent study, we have preparedcore-crosslinked pH-sensitive degradable micelles from PEG-b-poly(2,4,6-trimethoxybenzylidenepentaerythritol carbonate-co-acryloylcarbonate) [PEG-b-P(TMBPEC-co-AC)], which combined pH-sensitiveTMBPEC units and acryloyl-functionalized carbonate (AC) units in thehydrophobe (Fig. 5) [112]. The resulting micelles were efficientlycrosslinked by UV irradiation. The in vitro release studies showed thatPTX leakage from core-crosslinked PEG-b-P(TMBPEC-co-AC) micelleswas minimal at pH 7.4 even at low micelle concentrations, while fastPTX release was observed at endosomal pH due to hydrolysis of acetalbonds [44]. MTT assays revealed that PTX-loaded crosslinked pH-sensitive degradable micelles retained high antitumor activity compara-ble to PTX-loaded non-crosslinked counterparts, supporting efficientdrug release from PTX-loaded crosslinked micelles inside tumor cells.These core-crosslinked pH-responsive biodegradable micelles have
Table 5Functional nanocarriers based on halogen or azido-containing polycarbonates and copolymers
elegantly addressed the extracellular stability versus intracellular drugrelease dilemma of micellar anticancer drugs.
Alkene-functionalized polycarbonates are amenable to the “thiol-ene”click reaction, which proceeds either via the free radical addition of thiolsto carbon–carbon double bonds or via the Michael-addition of thiols toelectron-deficient carbon–carbon double bonds. Huang et al. reportedthat allyl-functionalized PEG-b-P(LLA-co-MAC) copolymer followingmodification with thioglycerol via the thiol-ene reaction and conjugationwith nucleobases (adenine and thymine) yielded PEG-b-P(LLA-co-MAC/adenine) and PEG-b-P(LLA-co-MAC/thymine) copolymers [113,114].PEG-b-P(LLA-co-MAC/thymine) copolymers could form stable nanoparti-cles by the addition of 9-hexadecyladenine (A-C16) due to the formationof hydrogen bonds [113]. The in vitroDOX release profile showed that theDOX release rate at pH 7.4 decreased with increasing A-C16 contents andfaster drug release was observed at an acidic pH of 5.0. In a followingstudy, PEG-b-P(LLA-co-MAC/adenine) and PEG-b-P(LLA-co-MAC/thymine) copolymers were found to form physically core-crosslinkedmicelles (Table 4), which had a lower CMC due to complementary mul-tiple hydrogen bonding interaction between adenine and thymine[114]. The in vitro release studies showed that DOX release was signifi-cantly restricted at pH 7.4 but enhanced at pH 5.0.
Ji et al. prepared a pH-sensitive polymeric prodrug, poly(5-methyl-5-allyloxycarbonyl-1,3-dioxan-2-one)-g-(12-acryloyloxydodecyl phosphorylcholine-co-6-maleimidocaproyl-doxorubicin)[PMAC-g-(ADPC-co-Mal-DOX)], by combining ROP and “thiol-ene” re-action [115]. Faster drug release was observed at endosomal pH(pH 5.0) than at physiological pH due to acid-triggered cleavage ofhydrazone linker. This prodrug exhibited high cytotoxicity againstHepG2 cells and HeLa cells. We synthesized poly(ε-caprolactone)-g-poly(2-hydroxyethyl methacrylate) (PCL-g-PHEMA) graft copolymersby combining ROP and reversible addition-fragmentation chain transfer(RAFT) polymerization, in which PCLmacro-RAFT agent was obtained bymodification of poly(caprolactone-co-acryloyl carbonate) [P(CL-co-AC)]copolymers with cysteamine via Michael-addition followed by couplingwith 4-cyanopentanoic acid dithionaphthalenoate (CPADN) [116]. Thesegraft polymeric micelles had a relatively low CMC and showed a pH-sensitive DOX release behavior. We also developed membrane-ionizablepolymersomes from cysteamine and mercaptopropionic acid-modifiedPEG-b-poly(trimethylene carbonate-co-acryloyl) [PEG-b-P(TMC-co-AC)]copolymers [117]. These polymersomes enabled highly efficient loadingof proteins as well as rapid protein release at endosomal pH. Very recent-ly, in situ forming reduction-sensitive degradable nanogelswere preparedfrom PEG-b-poly(2-(hydroxyethyl) methacrylate-co-acryloyl carbonate)[PEG-b-P(HEMA-co-AC)] block copolymers using cystamine as acrosslinker [118]. FITC-labeled cytochrome C (FITC-CC) could be readilyloaded into nanogels with remarkable loading efficiencies (up to 98.2%)and loading contents (up to 48.2 wt.%). The in vitro release of FITC-CCwasminimal under physiological conditions but significantly enhancedin the presence of 10 mM DTT with about 96.8% of FITC-CC released in22 h.
He et al. conjugated low molecular weight PEI (Mn = 423, 800 and1800 g/mol) onto poly(5-methyl-5-allyloxycarbonyl-1,3-dioxan-2-one) (PMAC) polymers to yield cationic biodegradable PMAC-g-PEIgraft copolymers for gene transfection [119,120]. The in vitro
.
Special advantages Refs.
Combination of ROP and “click reaction” [126,127]High drug loading capability [128]
PTX Combination of ROP and micro-wave assisted click reaction [129]Efficient gene delivery [130]Low cytotoxicity and efficient gene delivery [131]
Fig. 6. Proposed mechanism of DNA condensation by (a) PEG-b-cationic polycarbonate diblock copolymer and (b) cationic polycarbonate-b-PEG-b-cationic polycarbonate triblockcopolymer. [131], Copyright 2011. Reproduced with permission from Wiley-VCH Verlag GmbH & Co. KGaA.
408 W. Chen et al. / Journal of Controlled Release 190 (2014) 398–414
experiments using 293 T cells demonstrated that PMAC-g-PEI hadmuchlower cytotoxicity and a 3–10 fold higher transfection efficacy than 25 kPEI [119]. To probe the possible mechanism of PMAC-g-PEI mediatedtransfection, slowly degradable dimethyl carbonate (DTC) units wereintroduced into the backbone to tailor the degradation rate [120]. Theresults showed that graft polymer with the highest DTC content (upto 45 mol%) possessed the highest transfection activity and cell-compatibility, likely due to its balanced degradation rate for protectingDNA from degradation and releasing DNA inside the cells. Recently,they also conjugated DOX to PEG-b-poly (5-methyl-5-allyloxycarbonyl-1,3-dioxan-2-one-co-dimethyl carbonate) [PEG-b-P(ATMC-co-DTC)] viathe hydrazone liker for pH-triggered intercellular drug release in HeLacells [121].
6. Functional nanocarriers based on halogen or azido-containingpolycarbonates and copolymers
Halogen or azido-containing polycarbonates and copolymers can beprepared by ROP of halogen or azide-functionalized cyclic carbonates[76,122–125]. Halogen-functional polycarbonates can be modifiedwith sodium azide (NaN3) in DMF at various temperatures to obtainazido-functional polycarbonates (Scheme 6). For example, Shen et al.grafted PEG onto poly(5,5-dibromomethyl trimethylene carbonate-co-caprolactone) [P(DBTC-co-CL)] copolymers by converting bromo intoazido groups followed by “click” reaction with alkyne-terminated PEG
Table 6Functional nanocarriers based on sugar-containing polycarbonates and copolymers.
via Huisgen 1,3-dipolar cycloaddition [126]. In a following study, well-defined ibuprofen-grafted seven-arm amphiphilic star (P(DBTC-co-CL)-b-PEG)s copolymers were prepared based on β-cyclodextrin (β-CD) core by combining controlled ROP, esterification coupling reactionsand “click” reactions [127].
Azido-functional polycarbonates and copolymers can facilely besynthesized via ROP of azido-functional cyclic carbonate monomers.Zhuo et al. prepared amphiphilic PEG-b-poly[(dimethyl trimethylenecarbonate-co-2,2-bis(azidomethyl) trimethylene carbonate)-g-palmitate] [PEG-b-P(DTC-co-ADTC-g-Pal)] block copolymers by ROPof DTCmonomer and ADTCmonomerwith PEG as an initiator, followedby the click reaction between propargyl Pal and azido groups on thepolymer chains [128]. The self-assembled biodegradable micelleswere rather stable, had a high drug-loading capacity, and displayed asustained release of methotrexate (Table 5). Jing and coworkers report-ed that azido-carrying biodegradable polymers could be functionalizedwith alkynyl compounds such as anticancer drugs like gemcitabine andPTX or fluorescent dye like RhB viamicro-wave assisted click chemistry[129]. All the polymer–drug conjugates exhibited a pronounced antitu-mor activity against SKOV-3 and HeLa cell lines.
Halogen-functionalized polycarbonates and copolymers can also bemodified with tertiary amines such as trimethylamine or N,N,N′,N′-tetramethylethylenediamine (TMEDA), to provide cationic degradablepolymers for gene delivery. For example, Yang and coworkers reportedthat halide-functionalized polycarbonates following modification with
Tumor-targetability and efficient gene delivery [140]
O
R
PTMC-OHO O m
O
O nO
PTMC-b-P(MTC-sugar)MTC-sugar
O
OHO
HO OH
O
HO
O
HO
HO
OHOH
O
R':
O
Deprotection
HCOOH/H2O
O
R'
galactoseglucose
OHHO
O
OH OH
mannose
NH
NN
NNH
NH
HNNH2
n
O
O
O
O
O
O
O O
O O
O O
branched PEI-10 k
Deprotection
1 M HCl
OHO
O
O
O
OHOHHO
HO
O
HO
O
O
OOH
OH
OH
OH
O O
MTC-mannosePEI-g-(MTC-mannose)
(1)
(2)
Scheme 7. Synthesis of sugar-functionalized polycarbonates and copolymers.
409W. Chen et al. / Journal of Controlled Release 190 (2014) 398–414
bis-tertiary amines showed relatively high gene expression in HepG2,HEK293, MCF-7 and 4T1 cell lines even in the presence of serum[130]. In a further study, they found that cationic polycarbonate-b-PEG-b-cationic polycarbonate triblock copolymers exhibited the mostfavorable physicochemical (i.e., DNA binding, size, zeta-potential, andin vitro stability) and biological (i.e., cellular uptake and luciferase re-porter gene expression) properties, as compared to PEG-b-cationicpolycarbonate diblock copolymers and cationic polycarbonate controls(Fig. 6) [131]. Importantly, these cationic polycarbonate/DNA com-plexes were all shown biocompatible, inducing minimal cytotoxicityand hemolysis.
7. Functional nanocarriers based on sugar-containingpolycarbonates and copolymers
Carbohydrates are usually biocompatible and often can be degradedin the body. Carbohydrates are also able to have specific interactionswith proteins (lectins) on the cell surface [132]. Introduction of carbohy-drates into polycarbonate backbones or side chains presents an attractivemethod for targeted drug delivery (Table 6). As the hydroxyl groupsin sugar-functional cyclic carbonates are incompatible with the ring-opening process, all the hydroxyl groups need to be protected[133–137]. Yang and coworkers prepared amphiphilic glycopolymer-poly(trimethylene carbonate) block copolymers by a multi-stepprocedure, starting with the synthesis of protected sugar-containing [D-glucose, D-galactose, and D-mannose] cyclic carbonate monomers(Scheme 7), followed by sequential ring-opening copolymerizationof sugar-containing carbonate monomers and TMC monomer, anddeprotection using formic acid [138]. Self-assembled glycopolymer mi-celles with a high density of galactose in the shell displayed strong inter-action with ASGP-R positive HepG2 liver cancer cells. DOX-loadedgalactose-containing micelles showed a higher toxicity for HepG2 cellsthan for ASGP-R negative HEK293 cells. Yang and coworkers also
modified PEI with mannose-functionalized carbonate for the delivery ofplasmid encoding granzyme B (GrB) inhibitor, proteinase inhibitor-9(PI-9), to prevent GrB-induced apoptosis of the target cells [139]. PEIwith partial primary amine groups substituted by the carbohydrate hadsimilar gene binding ability to the parent PEI. Interestingly, they showedalmost 100% transfection efficiency of a GFP-reporter plasmid inHEK293T human embryonic kidney cells (Table 6). Very recently, well-defined galactose-functionalized cationic polycarbonate diblock copoly-mers (designated as Gal-APC) containing quaternary and tertiary aminesfor DNA binding and endosomal buffering were developed for selectivegene delivery to ASGP-R-overexpressing hepatocytes. Gal-APC/DNAcomplexes mediated significantly higher gene expression in HepG2cells, as compared to glucose-functionalized copolymer/DNA complexes[140].
8. Functional nanocarriers based on polycarbonates and copolymerscontaining other functional groups
Polycarbonates bearing several other functionalities such as PEG,cholesterol, 2,4-dinitrophenylthioether, and pyridyl disulfide havealso been developed and investigated for controlled drug delivery(Scheme 8, Table 7). For example, well-defined amphiphilic PCL-b-poly(DTC-co-cyclic carbonate-terminated oligo(ethylene glycol)) [PCL-b-P(DTC-co-(MTC-PEG))] block copolymers with PEG methyl etherpendant chains were facilely prepared by ROP of MTC-PEG monomer[141]. PTMC copolymers with PEG in the main chain or at the sidechain were found to show temperature sensitivity [142,143]. Yanget al. reported that block copolymers of PEG and polycarbonatesprepared from cyclic carbonate monomers derived from 2,2-bis(methylol)propionic acid (bis-MPA) with hydrophilic (MTC-PEG)and hydrophobic (MTC-C2 and MTC-C12) groups displayed differentlower critical solution temperatures (LCST), depending on theMn of hy-drophilic PEG chains and composition of copolymers [144]. PTX-loaded
Scheme 8. Synthesis of polycarbonates and copolymers functionalized with variousgroups such as PEG, cholesterol, 2,4-dinitrophenylthioether, and pyridyl disulfide.
410 W. Chen et al. / Journal of Controlled Release 190 (2014) 398–414
micelles killed HepG2 human liver carcinoma cells more efficiently at37 °C (mimicking the body temperature) as compared to free PTX andPTX-loaded micelles at temperatures below the LCST.
In a recent report by Yang et al., a novel cyclic carbonate with apendant cholesterol group (MTC-Chol), was homopolymerized (DP11) or copolymerized with TMC using PEG as a macroinitiator toprepare biodegradable micelles for controlled release of PTX [145].The Chol-containing micelles (molar ratio of Chol/TMC units of 1/3)had a relatively high PTX loading capacity (15 wt.%) due to hydrophobicinteraction with cholesterol (Table 7). Near-infrared fluorescence(NIRF) imaging studies using a 4T1 Balb/c mouse breast cancermodel showed that these micelles allowed effective passive targeting,and were preferably accumulated in tumor tissue with limiteddistribution to healthy organs. They also found that amphiphiliccholesterol-functionalized PEG113-b-poly(2-(5-methyl-2-oxo-1,3-dioxane-5-carboxyloyloxy)ethyl carbamate)n [PEG113-b-P(MTC-
Table 7Functional nanocarriers based on polycarbonates and copolymers containing other functional
Polymers Nanosystems Mo
Type Size (nm)
P(MTCPEG-co-MTCC2 or C12) Micelle 16–60 PTPEG-b-P(TMC-co-MTCChol) Micelle 80–125 PTPEG-b-P(LA-co-MTCSH) Micelle 97–145 DOPCL-g-SS-PEG Micelle 110–120 DOPCL-g-SS-LBA Nanoparticle 80 DO
Chol)n] block copolymers had a unique self-assembly behavior in aque-ous solution, showing disk-like micelles (n = 4) and structures with astacked-disk-like morphology (n = 11) [146].
The S\S bonds are relatively stable under physiological conditionsas well as in extracellular tissues, while rapid degradation takes placeintracellularly due to presence of a higher GSH concentration [147].Engler et al. designed and synthesized polycarbonate-based brush poly-mers with detachable, disulfide-linked, hydroxyl-terminated pendantside chains by ROP of tritylthio ethanol-functionalized carbonate(MTC-OTrThiol) and BTMCC, deprotection of thiol groups and thiol–disulfide exchange with 2-(2-pyridyldithio)ethanol [148]. Jing et al. ob-tained amphiphilic biodegradable copolymers containing free thiolgroups after deprotection of 2,4-dinitrophenyl-protected carbonateunits (MTC-DNPT) [149]. The copolymers could be self-assembled intomicelles in aqueous solution and crosslinked by oxidation of free thiolgroups in the micellar core to enhance the stability (Table 7). Thein vitro release behavior indicated that DOX release from micelles wasinhibited bymicelle core-crosslinking,while the release ratewas greatlyaccelerated by treatmentwith GSH at a level analogous to that in tumorcells.We recently prepared a new cyclic carbonatemonomer containingpyridyl disulfide group (PDSC), with which poly(ε-caprolactone)-g-SS-PEG (PCL-g-SS-PEG) graft copolymers were obtained through copoly-merization with ε-CL followed by thiol–disulfide exchange reactionwith thiolated PEG [150]. PCL-g-SS-PEG graft copolymers self-assembled into robust reduction-sensitive micelles with a low CMC inwater. The in vitro release studies revealed that DOX release wastriggered under a reductive condition (Fig. 7). MTT assays showedthat DOX-loaded PCL-g-SS-PEG micelles induced pronounced anti-tumor activity to HeLa cells with an IC50 close to that for free DOX. In amore recent report, PCL-graft-SS-lactobionic acid (PCL-g-SS-LBA)glyco-nanoparticles (SS-GNs) with sheddable saccharide shells weredeveloped as a unique and potent platform for hepatoma-targeting de-livery of DOX [151]. Theflow cytometry and confocalmicroscopy obser-vations indicated that SS-GNs could be efficiently taken up by ASGP-Roverexpressing HepG2 cells likely via a receptor-mediated endocytosismechanism, and DOX was released into the nuclei of cells followingonly 4 h incubation due to the fast cleavage of S\S bonds at intracellularreductive conditions.
9. Conclusions
Thepast several years havewitnessed a rapid development of biode-gradable nanocarriers based on functional polycarbonates and copoly-mers for controlled drug and gene delivery. It is interesting to notethat cyclic carbonate monomers with various functional groups can bereadily prepared and undergo ring-opening polymerization undermild conditions. Moreover, functional cyclic carbonate monomers canalso copolymerize with different cyclic ester or carbonate monomers,which provide a versatile access to advanced biodegradable nanocarriers.For example, based on designed cyclic carbonate monomers, functionalnanocarriers with bioresponsivity (e.g. pH and redox-sensitivity),thermosensitivity, improved stability (through chemical crosslinking,hydrogen bonding, etc.), and chemical conjugation ability (conjugationof therapeutic agents, targeting ligands, fluorescence probes, etc.) havebeen developed for in vitro and in vivo drug delivery. Furthermore,
groups.
del drugs Special advantages Refs.
X Thermal-sensitivity [144]X High drug loading capability [145]X S-S crosslinking and reduction-sensitivity [149]X Reduction-sensitivity [150]X Reduction-sensitivity and tumor targetability [151]
SH
SH
HS
HS
HSSH
SH HSIntracellular reductive
environment
Anticancerdrug ( )
OSH
n
OO
OO O
SSN
x y n
PDSC
-CLε
ROP
P(CL-co-PDSC)
Thiol-disulfideexchange reaction
PEG-SH
Drug-loaded shell-sheddablebiodegradable micelle
Reduction-triggered disassociationand drug release
Self-assembly
+O O
O
SS
N
O
O
PCL-g-SS-PEG
OO
OO O
Sx y n
SHNOO
Oz
Fig. 7. Synthesis of functional PCL containing pendant pyridyl disulfide groups and facile access to reduction-sensitive biodegradable graft copolymermicelles for intracellular drug release.[150], Copyright 2013. Reproduced with permission from the American Chemical Society.
411W. Chen et al. / Journal of Controlled Release 190 (2014) 398–414
cationic polycarbonates and copolymers have shown a great potentialas vectors for gene delivery. It should be noted, nevertheless, that allfunctional polycarbonates have been assumed to possess excellentbiocompatibility and biodegradability as PTMC, which is perhaps nottrue. Reports about the in vivo degradation mechanism of these func-tional polycarbonates are lacking. In order to progress to clinical appli-cations, systemic studies on the in vivo fate and safety of functionalpolycarbonates and copolymers should be carried out. We envisagethat these functional polycarbonates and copolymers will play a greatrole in constructing innovative and efficient nanosystems for targetedcancer chemotherapy.
Acknowledgment
This workwas supported by theNational Natural Science Foundationof China (NSFC 51003070, 51103093, 51173126, and 51273139), theNational Science Fund for Distinguished Young Scholars (51225302),and a Project Funded by the Priority Academic Program Developmentof Jiangsu Higher Education Institutions.
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