Fibrin nanoconstructs: a novel processing method and their use as controlled delivery agents This article has been downloaded from IOPscience. Please scroll down to see the full text article. 2012 Nanotechnology 23 095102 (http://iopscience.iop.org/0957-4484/23/9/095102) Download details: IP Address: 146.229.56.102 The article was downloaded on 02/08/2013 at 13:56 Please note that terms and conditions apply. View the table of contents for this issue, or go to the journal homepage for more Home Search Collections Journals About Contact us My IOPscience
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Fibrin nanoconstructs: a novel processing method and their use as controlled delivery agents
This article has been downloaded from IOPscience. Please scroll down to see the full text article.
2012 Nanotechnology 23 095102
(http://iopscience.iop.org/0957-4484/23/9/095102)
Download details:
IP Address: 146.229.56.102
The article was downloaded on 02/08/2013 at 13:56
Please note that terms and conditions apply.
View the table of contents for this issue, or go to the journal homepage for more
Home Search Collections Journals About Contact us My IOPscience
Fibrin nanoconstructs: a novel processingmethod and their use as controlleddelivery agents
G Praveen, P R Sreerekha, Deepthy Menon, Shantikumar V Nair andKrishna Prasad Chennazhi
Amrita Center for Nanosciences and Molecular Medicine, Amrita Institute of Medical Sciences andResearch Centre, Amrita Vishwa Vidyapeetham University, Kochi, Kerala 682041, India
Received 19 August 2011, in final form 10 January 2012Published 6 February 2012Online at stacks.iop.org/Nano/23/095102
AbstractFibrin nanoconstructs (FNCs) were prepared through a modified water-in-oilemulsification–diffusion route without the use of any surfactants, resulting in a high yieldsynthesis of fibrin nanotubes (FNTs) and fibrin nanoparticles (FNPs). The fibrinnanoconstructs formed an aligned structure with self-assembled nanotubes with closed headsthat eventually formed spherical nanoparticles of size ∼250 nm. The nanotubes were typically∼700 nm long and 150–300 nm in diameter, with a wall thickness of ∼50 nm and porediameter of about 150–250 nm. These constructs showed high stability against aggregationindicated by a zeta potential of −44 mV and an excellent temperature stability upto 200 ◦C.Furthermore, they were found to be enzymatically degradable, thereby precluding any longterm toxicity effects. These unique fibrin nanostructures were analyzed for their ability todeliver tacrolimus, an immunosuppressive drug that is used widely to prevent the initial phaseof tissue rejection during allogenic transplantation surgeries. Upon conjugation withtacrolimus, a drug encapsulation efficiency of 66% was achieved, with the in vitro releasestudies in PBS depicting a sustained and complete drug release over a period of one week atthe physiological pH of 7.4. At a more acidic pH, the drug release was very slow, suggestingtheir potential for oral–intestinal drug administration as well. The in vivo drug absorption ratesanalyzed in Sprague Dawley rats further confirmed the sustained release pattern of tacrolimusfor both oral and parenteral delivery routes. The novel fibrin nanoconstructs developed using agreen chemistry approach thus proved to be excellent biodegradable nanocarriers for oral aswell as parenteral administrations, with remarkable potential also for delivering specificgrowth factors in tissue engineering scaffolds.
(Some figures may appear in colour only in the online journal)
1. Introduction
Nanoparticles have drawn increasing interest in the biomed-ical arena owing to their ability to deliver drugs at optimaldoses, often resulting in increased therapeutic efficacy ofthe drug, weakened side effects, and improved patient com-pliance. However, important considerations while designingsuch nanoparticle based drug delivery agents include seriousconcerns about its nanotoxicity aspects such as cytotoxicity,
genotoxicity, and blood compatibility [1]. Biodegradability,bioactivity, drug loading capacity as well as the drug releasekinetics of the nanoconstructs are also of prime concernin developing novel nanocarriers. Poly(lactide-co-glycolide)represents the only synthetic drug delivery agent approvedby the US-FDA for drug delivery applications this far [2].Other natural materials that have been used include starch,proteins, lectins and chitins [3–6]. Fibrin, a bio-polymernaturally synthesized during the coagulation cascade is a
Figure 1. Schematic representation of the surfactant-free water-in-oil emulsification–diffusion system designed for preparing fibrinnanoconstructs with a chart of the main steps involved in the preparation.
less exploited natural material, and is the body’s choice ofa natural agent for the absorption and delivery of severalfactors. It also serves as an ideal substrate for cell attachment,proliferation, extracellular matrix formation, eventual tissueregeneration, and can easily be degraded as well as toleratedby the biological milieu [7–10]. The promising applicationsand research progress using fibrin in tissue engineering anddrug delivery to date have been recently reviewed by Breenet al [11]. An approach of synthesizing fibrin nanoparticleswas previously reported using a process wherein fibrinogenwas first mixed with thrombin and then introduced to an oilemulsion to obtain fibrin microbeads of varying size from 60to 180 µm which were then subsequently homogenized toderive a very small fraction of fibrin nanoparticles [12]. In thisstudy, we are presenting a new approach of surfactant-free,water-in-oil emulsification–diffusion technique that enablesus to synthesize fibrin nanoconstructs (FNCs) with high yieldand devoid of having any microstructures. The applicabilityof these constructs for oral and parenteral drug deliverywas verified by loading an immunosuppressive drug, namely,tacrolimus. The in vitro as well as in vivo efficacy ofdrug loaded FNCs was evaluated in detail. The samemethodology was tested to incorporate angiogenic growthfactor and henceforth to report its prospective value in tissueengineering applications as a scaffold component aiding tissueregeneration when used for the controlled delivery of drugsand growth factors, or their combinations.
2. Materials and methods
2.1. Isolation of fibrinogen from frozen blood plasma
Fibrinogen was isolated from human blood plasma by afreeze–thaw cycle [13]. Briefly, human plasma bags wasobtained from the hospital blood bank of Amrita Institute ofMedical Sciences (AIMS) through prior written consent andwas first frozen at −20 ◦C for 24 h and then thawed at 4 ◦Cfor 18 h. Plasma was centrifuged at 6500g for 20 min usinga refrigerated centrifuge (Hermle—Z36-HK, Germany) at
4 ◦C. The precipitate was dissolved in 0.9% sodium chloridesolution, which was then frozen at −20 ◦C for 2 h andlyophilized for 18 h using a freeze-drier (Alpha 2-4 LD plusChrist, Germany) to obtain fibrinogen powder, enriched by thecoagulation component FXIIIa.
2.2. Synthesis of fibrin nanoconstructs (FNCs) and itsconjugation with tacrolimus and VEGF
A surfactant-free water-in-oil emulsification–diffusion systemwas designed to induce thrombin–FXIIIa mediated crosslinking of fibrin molecules. The basis of this new approachinvolves the simultaneous administration of fibrinogen andthrombin taken separately in individual applicators, allowingits interaction only in the pre-heated vegetable oil system(figure 1). Another aspect of this method that is differentfrom previously reported approaches is that the particles werecollected at the interface of oil and water by density gradientseparation. This was followed by probe sonication thatenabled good control over both the shape as well as size of theresultant nanoconstructs. In brief, 5 ml aqueous suspensionsof both fibrinogen–FXIIIa cryoprecipitate and thrombin wereinstantaneously injected into 40 ml of purified vegetable oil.These aqueous suspensions were allowed to crosslink andemulsify in the oil phase, which was kept under constantmagnetic stirring at 400 rpm and maintained at a temperatureof 70–80 ◦C. The system was allowed to equilibrate understirring for 6–8 h, wherein the cross linking of fibrin clotand its uniform dispersion in oil occurred. The nanoconstructsthus formed in the emulsion were then centrifuged at10 000 rpm for 15 min. This resulted in the formation ofa density gradient layer of nanoconstructs at the oil–waterinterface, which was pooled up and harvested from theemulsion. The resultant constructs were further redispersedin water and probe sonicated for 20 min to attain a uniformnanosize distribution and preserved after lyophilizing for 48 h.For synthesizing tacrolimus conjugated fibrin nanoconstructs(T-FNCs), tacrolimus (as monohydrate) was purchased fromConcord Biotech Limited (Ahmedabad, India). 1 mg oftacrolimus was dissolved in 1 ml of ethanol:PBS (1:2)
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Nanotechnology 23 (2012) 095102 G Praveen et al
solution and mixed with 4 ml of aqueous fibrinogen–FXIIIacryoprecipitate. For synthesizing VEGF conjugated fibrinnanoconstructs (V-FNCs), recombinant human VEGF waspurchased from PeproTech Inc. (Rocky Hill, USA). Sampleswere prepared with the addition of 1 µg VEGF to 1 mlof Hank’s balanced salt solution and mixed with 4 mlaqueous suspension of fibrinogen–FXIIIa cryoprecipitate.The procedures further to this step were identical to thoseadopted for the preparation of FNCs as explained above.The rheological behavior of the oil systems (vegetable oilssuch as sesame, gingelly, and coconut oils) used for thesynthesis was evaluated by measuring the viscosity as afunction of temperature in the range 50–80 ◦C using adigitalized viscometer (DV-II Pro., Brookfield, USA). Whilesynthesizing V-FNCs, additional heed was taken to confinethe experimental temperature between 45 and 50 ◦C.
2.3. Physicochemical characterization of fibrinnanoconstructs
2.3.1. Hydrodynamic particle size and surface chargeanalysis. The hydrodynamic particle size distribution ofFNCs was analyzed using the technique of dynamic lightscattering (DLS) by measuring the intensity of scatteredlight as the FNCs underwent Brownian motion. Thesamples suspended in MilliQ water were analyzed usingNicompTM 380 ZLS-ZP/Particle Sizer (Santa Barbara, USA)with a laser source emitting at 632.8 nm, and the scatteredintensity measured at an angle of 90◦. The intensity-weightedGaussian distribution was recorded as the average of threemeasurements. Surface charge and thereby the stabilityof the same system was analyzed from its zeta potentialmeasurements using the Nicomp Zetasizer.
2.3.2. Morphological analysis of FNCs through SEM andAFM. The size as well as the surface topographyof FNCs and T-FNCs was analyzed by scanning electronmicroscopy (SEM) and atomic force microscopy (AFM).For SEM analysis, freeze-dried nanoconstructs redispersed inMilliQ water were dropped on aluminum stub and sputtercoated with platinum using an automatic fine platinum coater(JEOL JFC-1600) at 10 mA for 120 s before imaging underSEM (JEOL-JSM-6490LA). For AFM analysis, 1 mg ml−1
suspension of freeze-dried nanoconstructs was prepared anddrop cast over an atomically flat mica surface and air-dried.The atomic force microscopic images were acquired using anAFM (JEOL-SPM-5200) in tapping mode at a scan size of2 µm× 2 µm at a scan rate of 333.33 µs.
2.3.3. Spectroscopic analysis. FNCs were further evaluatedfor their functional characteristics using Fourier transforminfrared (FTIR) spectroscopy. For FTIR analysis, 2 mg offreeze-dried nanoconstructs were pelletized with 175 mgKBr and the spectrum recorded in the frequency range650–2100 cm−1 using an RX1-FT-IR spectrometer (PerkinElmer, USA).
2.3.4. Thermogravimetric analysis. Thermal stability ofbare tacrolimus, FNCs, and T-FNCs were analyzed by thethermogravimetric analysis (TGA) using the Exstar TG/DTA6200 system (SII NanoTechnology Inc., USA). 2 mg each ofthe samples was subjected to thermal degradation studies inthe temperature range spanning 35–450 ◦C at a progressiverate of 10 ◦C min−1 under inert nitrogen environment.
2.3.5. Physicochemical staining (PTAH). Mallory’sphosphotungstic acid hematoxylin (PTAH) staining techniquewas adopted to specifically stain the core fibrin component ofthe nanoconstructs. In brief, the freeze-dried nanoconstructswere dehydrated in 80% ethanol and pre-stained with eosinfor 5 min. After washing with double distilled water theparticles were stained with PTAH, incubated at 60 ◦C for30 min and viewed under the bright field mode of a fluorescentmicroscope (Olympus-BX-51).
2.3.6. In vitro degradation and SDS-PAGE analysis ofnanoconstructs. The in vitro enzymatic degradation ofFNCs was performed by incubating freeze-dried nanoparticles(5 mg ml−1) in salined buffer containing 60 µg ml−1
For evaluating the effect of FNCs on cell viability, a directproliferation assay was performed using human umbilicalcord blood derived mesenchymal stem cells (hMSCs).Umbilical cord blood was collected from the gynecologydepartment after obtaining the prior ethical consent of theinstitutional body of Amrita Institute of Medical Sciences.The isolation of hMSCs was performed as per the establishedprotocol [14] and the healthy cells were maintained upto passage 3 and taken further for experiments. In brief,about 10 000 cells/well were seeded in a 24-well tissueculture polystyrene and incubated for 72 h with varied
The encapsulation efficiency and in vitro release rate oftacrolimus were analyzed at a wavelength of 213 nm atpredetermined time intervals of 0, 1, 2, 4, 6, 8, 12, 24,48, 96, 120, 144, and 168 h using an HPLC system(LC-2010 A HT, Shimadzu) under the following conditions:mobile phase—100% acetonitrile, column—luna 5u C18,250 mm × 4.6 mm (phenomenex), flow rate—0.5 ml min−1.A standard calibration curve was initially generated fortacrolimus concentrations between 6.25 and 500 µg ml−1
that yielded a good linear correlation (R2= 0.995). Partial
medium replacement was performed after each analysis byadding 100 µl of fresh buffered solutions to the suspension.The suspension was rear filtered each time using the filterdevice after adding 100µl of fresh buffered solutions and keptback for incubation.
2.6. In vivo drug absorption studies in rats
Adult Sprague Dawley rats (N = 12) weighing 210–240 gwere used for the in vivo studies designed to look at thedrug absorption of tacrolimus loaded FNCs administeredthrough oral and intravenous routes. The rats were housedin separate cages and kept at a controlled temperature (23 ±2 ◦C) and relative humidity (45 ± 5%). The approval ofthe Institutional Ethical Committee (IEC) was acquired for
performing the in vivo studies in rats and NIH guidelineswere followed for the care and use of laboratory animals.The animals were exposed to a 12 h light/dark cycle, fastedovernight and had free access to sterile drinking water. Theanimals were divided into four groups (A, B, C, and D) ofthree each. Drug administration was performed through oralgavage and tail vein injection. Rats of Group A were suppliedwith bare tacrolimus drug suspension 1 mg/rat (4 mg kg−1)through the oral route, while Group B was administered withbare tacrolimus 1 mg/rat (4 mg kg−1) through tail veininjection. Rats of Groups C and D were given an emulsionof T-FNCs containing similar concentrations of tacrolimus asabove via oral and tail vein injection respectively. Furtherto the administration, blood samples (200 µl) were drawnfrom the rats through retro-orbital sinus bleeding usingnon-heparanized capillary tubes. The blood was collected in0.5 ml microcentrifuge tubes at predetermined time intervals(0, 30, 60, 120, 240, 360, and 1440 min.). Serum fromthe blood samples was separated out by centrifuging at12 000 rpm for 20 min. The separated serum was then mixedwith equal volume of 80% methanol for precipitating serumproteins. The samples were then analyzed for their drugcontent using HPLC as detailed above. Total drug absorption(mg/wholeblood) was calculated assuming a blood volume of15 ml per rat.
2.8. In vitro analysis of VEGF assisted cell proliferation andtube formation of HUVEC
In order to evaluate the VEGF assisted cell proliferationand patterned endothelialization induced among humanumbilical vein endothelial cells (HUVEC), cells were isolatedfrom the umbilical cord samples as per the establishedprotocol [15]. Umbilical cord was collected from the personswho underwent normal delivery at the gynecology departmentof AIMS with their prior written consent and also the ethicalclearance of the institutional body of AIMS. The detachedcells were washed in serum-free IMDM and resuspended incomplete IMDM (containing 20% fetal calf serum (GIBCO,Invitrogen), 100 U ml−1 pen-strep antibiotic solutions(GIBCO, Invitrogen, USA), and 150 µg ml−1 ECGF). TheHUVECs were grown on 2% gelatin (Sigma Aldrich, USA)coated tissue culture plates in complete IMDM in a humidified
of FNCs and V-FNCS and incubated at 37 ◦C. The mediachange was given every alternate day. The total cells per wellwere trypsinized and proliferation was evaluated at 24 h, onthe third, fifth, and seventh days.
For cell migration experiments, confluent HUVEC cellsin passage 3 having a typical cobblestone morphologywere seeded over a 3D macroporous scaffold environmentframed by casting 6% agarose embedded with 10 mgof freeze-dried V-FNCs. In brief, 6% Low EEO Agarose(Lonza Cologne,Germany) was dissolved in sterile PBS byboiling and lyophilized followed by gelling. Multi-tieredarchitecture with an approximate layer thickness of 25 µmwas molded over a flat glass surface. Freeze-dried V-FNCswere added to the agarose gel while it was cooled down bymoderate agitation resulting in the formation of a V-FNCrich zone (unpublished data). The effect of eluted VEGFon the patterned alignment of endothelial cells and tubeformation was studied over this zone. Cells were imaged onday 7 of the experiment using a confocal microscope TCSSP5-II (Leica, Germany). The cells were stained with Texasred-labeled anti-CD31 (Santa Cruz, USA) and cell nuclei werespecifically stained using DAPI (Invitrogen) fluorescenceexcited with a He–Ne laser at 594 nm and 350 respectively.
3.1. Preparation and characterization of fibrinnanoconstructs
It is well known that the surface morphology as well as sizedistribution of nanoparticles prepared through the emulsionroute is significantly influenced by the choice of the oilmedium and thereby its rheological properties [17]. Hence,in the present experiment to prepare fibrin nanoconstructsof desired size and morphology, we have carried out initialexperiments by changing the oil phase, oil–water ratio aswell as the temperature. Moreover, the use of vegetable oilsin this preparation would help to enhance the biocompatiblenature of FNCs to a considerable level. Thus, the preparationof FNCs was carried out using three selected vegetable oils(coconut, sesame, and sunflower oil) after assessing their
Figure 2. Dynamic light scattering based intensity-weighted sizedistribution of fibrin nanoconstructs depicting their uniformity andstability.
rheological behavior. By comparing the rheological behaviorsof the three different oil systems selected for the study, itwas noted that the viscosity of coconut oil is relatively stablefor changing temperatures, in comparison to sunflower andsesame oils used for the study. Consequently, we selectedcoconut oil for the synthesis of FNCs using the water-in-oilemulsion route.
The green emulsification route adopted for the synthesisof FNCs in this study has significant advantages in terms ofits size as well as stability in comparison to similar worksreported using alginate soft beads and fibrinogen [18, 19].For the preparation of fibrin nanoconstructs using coconutoil as the oil phase, no additional surfactants were used tostabilize the emulsion, making it a completely bio-friendlysynthesis route. Additional stability of this system can alsobe attributed to the strong ε-(γ -glutamyl) lysine covalentinteraction of activated fibrin residues formed in the presenceof two constitutive self-association sites in the γ -chain ofD-domains exposed during the thrombin mediated cleavage offibrinogen precursors [20]. Figure 2 depicts the hydrodynamicparticle size distribution of the FNCs with particles in therange of about 240 ± 5 nm. and the mean zeta potentialvalue was estimated to be of −44 mV for the colloidalsystem of FNCs suggesting its excellent stability, which canbe attributed to the stabilizing effect of negatively chargedtriglyceride ions exposed over the protein nanoparticles [21].
Morphology and surface topography analysis performedthrough SEM and AFM (figure 3) confirmed the presenceof monodispersed nanoparticles and a tubular protein array,together constituting the fibrin nanoconstructs. The proximalends of FNCs as evident from the SEM images (figures 3(a)and (b)) are self-assembled to form a tubular array and areclosed at the distal end with spherical fibrin nanoparticles(FNPs) with mean size scale of 240 ± 5 nm (figure 3(c)).A plausible reason for the formation of this particulararrangement of bead and tube array is the immediate crosslinking of precursor fibrinogen and thrombin moleculesduring the initial experimental phase itself while the systemis kept under stirring for about 6 h. It is assumed that,while the system is kept for stirring (about 6 h), the tubelike structures are eventually formed by the cross linking of
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Figure 3. Scanning electron microscopy images: ((a), (b)) angular and top views of fibrin nanoconstructs depicting the assembly of fibrinnanotubes; (c) AFM topography images of fibrin nanoconstructs.
Figure 4. FTIR spectroscopic comparison of fibrin nanoconstructsbefore and after conjugation with tacrolimus: (a) FNCs alone,(b) tacrolimus alone, and (c) tacrolimus conjugated FNCs.
unreacted fibrinogen mediated by factor XIII. However, theexact mechanism of the formation of fibrin nanotubes in thesystem is not completely unraveled.
In order to analyze the molecular integrity and toascertain the degree of drug conjugation that occurred whentacrolimus was incorporated into fibrin nanoconstructs, FTIRspectroscopic analysis was performed. The IR spectrum ofbare fibrin figure 4(a) exhibited typical amide bands at1650 cm−1 (amide I), 1550 cm−1 (amide II), and 1220 cm−1
(amide III) which are characteristic to this material [22].Several intense peaks were clearly evident in the FTIR
Figure 5. Thermal degradation profile of fibrin nanoconstructsevaluated using thermogravimetric analysis.
spectrum of bare tacrolimus figure 4(b). On conjugation oftacrolimus with fibrin nanoconstructs, the prominent peaks offibrin merged with tacrolimus peaks figure 4(c) and appearedwith reduced intensity, pointing to the drug conjugation.
The thermal degradation profile of T-FNCs clearlyexhibits a stable plateau in the degradation curve uptohigher temperatures (∼260 ◦C) indicating a high degree ofthermal stability exhibited by fibrin based nanoconstructs afterconjugation with the drug (figure 5).
For visualizing the physiologically unaltered fibrincomponent of the T-FNCs, histochemical staining using phos-photungstic acid and hematoxylin (PTAH) was employed. It
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Nanotechnology 23 (2012) 095102 G Praveen et al
Figure 6. Optical micrograph of histochemical staining of T-FNCsusing phosphotungstic acid and hematoxylin (PTAH) thatspecifically imparts a dense blue color to the fibrin component(magnification ×60).
is already reported that the binding of PTAH by fibrin isdue to hydrogen bonding occurring between two phenolic-OHgroups of each hematein ligand chelated within the suitablyspaced sites of amino peptides [23]. The evenly spacedphenolic-OH groups of chelated ligand specifically impart ablue color to the unaltered amino peptides of fibrin, as evidentfrom figure 6, thereby proving the unaltered state of fibrinwithin the T-FNCs.
The potential use of fibrin based microbeads (FMBs)in adult stem cell based techniques was reported earlier byGurevich et al [24] particularly to separate mesenchymalstem cells (MSCs) from a much wider spectrum ofmononuclear cell sources. Hence, for evaluating the in vitrocell viability potential of fibrin based nanoconstructs, humanmesenchymal stem cells (hMSCs) were used in the presentinvestigation. The cells were incubated in direct contact withthe nanoconstructs and it was proved that upto 5 mg ml−1
suspension of FNCs and T-FNCs do not impart any notabletoxicity to the cells (figure 7) and thus prove to be idealcandidates for further biological experiments.
Figure 7. In vitro cell viability of bare and tacrolimus conjugatedfibrin nanoconstructs assessed through alamar blue assay usinghuman mesenchymal stem cells.
3.2. In vitro degradation and SDS-PAGE analysis ofnanoparticles
The in vitro degradation of FNCs was evaluated by theaddition of active MMP-3 and the enzymatic action offibrin was monitored spectrophotometrically over an extendedperiod of time. The specific cleaving pattern induced by activeMMP-3 and the molecular weight based filtration techniqueenabled this spectrophotometric evaluation and thus helpedin calculating the pace of enzymatic degradation impartedduring in vitro incubation. The UV–vis absorption patternof the filtered samples as shown in figure 8(a) recordedat 280 nm clearly indicated that a considerable numberof peptide residues are released after the eighth hour ofenzymatic hydrolysis of fibrin. The enzymatic degradationprofile obtained for T-FNCs was more or less similar tothat of FNCs, thereby indicating that drug loading has notaltered the degradation profile of the protein nanocarrier.Hence, it can be concluded that fibrin based nanoparticlescan be easily degraded and cleared off within 8–10 h whenadministrated into a biological system. The enzymaticallydegraded products were further examined by SDS-PAGE toresolve and identify the protein subunits specifically affectedby MMP-3 induced enzymatic hydrolysis [25]. Degradation
Figure 8. (a) In vitro enzymatic degradation profile and (b) specific cleaving pattern of fibrin nanoconstructs (lane-3) resolved aftertreatment with active matrix metalloprotease-3 (MMP-3).
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Nanotechnology 23 (2012) 095102 G Praveen et al
Figure 9. In vitro drug release profile of tacrolimus conjugatedfibrin nanoconstructs at pH 7.4 and pH 1.5. The values represent themean ± SD (n = 6) (p < 0.05, Anova).
products after 18 h of in vitro incubation appeared asintensified bands in electrolytic separation and were observedas fully resolved bands after 96 h. The hydrolysis of fibrinby active MMP-3 resulted in the degradation of the α-chains(240–245 kDa), while the β-chains (120 kDa), γ -chain(140 kDa), and the γ –γ dimer (140–145 kDa) appeared asdistorted smears indicating the high degree of unspecifiedcleavage that occurred due to the prolonged exposure toMMP-3 (figure 8(b)).
3.3. In vitro drug release experiments
The encapsulation efficiency of tacrolimus was initiallymeasured spectrophotometrically to be ∼66%. Further, thein vitro release pattern of tacrolimus in ethanol:PBS (1:2ratio) buffered solutions at (a) pH 7.4 and (b) pH 1.5was recorded as shown in (figure 9). Tacrolimus showshigh between- and within-subject pharmacokinetic variability.Due to its narrow therapeutic index, close drug monitoringprograms are required to optimize its efficacy and limitits toxicity [26]. Through this study, it was observed thatthe release profile of tacrolimus from T-FNCs is slowand sustained, with no initial burst release under normalphysiological pH. Nearly 97% of the drug was released overa period of seven days. However, in contrast, the releaseprofile obtained at pH 1.5 was considerably different, withonly about 30% of the drug released for the same duration,with negligible release rates for the initial durations upto6 h. This peculiar release pattern obtained at acidic pH canbe attributed to a possible differential degradation kineticsexhibited by fibrin at varying pH conditions as reportedearlier [27, 28]. The hardening of fibrin nanoconstructs uponexposure to dilute hydrochloric acid can be a plausible reasonthat prevents the release of tacrolimus from FNCs duringthe initial phase of the study. However, prolonged exposureof FNCs to the acid medium could result in denaturingof the cross linked fibrin matrix, eventually leading to apartial release of the conjugated drug from FNCs within thestipulated time (seven days). Thus, from our experimentalresults, it is evident that pH dependent microenvironmentscritically influence the release of tacrolimus conjugated withFNCs, depending on the degradability of fibrin at variedpH and the subsequent drug diffusion from the matrix.
Figure 10. In vivo drug absorption studies in SD rats administratedwith tacrolimus as bare and FNC conjugated formulations throughoral and tail vein routes. The results are expressed as mean ± SD(n = 6) (p < .05, Anova).
Henceforth, it is concluded that T-FNCs are pH sensitive withpotential applications for controlled drug release in bloodas well as intracellular acidic environments and the overallresults suggest that this nanodelivery system can be usedas a potential platform for intestinal transport of sensitivelipophilic molecules like tacrolimus that are P-gp substrates.This aspect was further analyzed through in vivo experimentsin SD rats, by administering T-FNCs via oral and intravenous(iv) routes.
3.4. In vivo drug absorption studies in rats
The concentration profiles of tacrolimus in rat blood serumafter oral and iv administration of bare tacrolimus and T-FNCsare represented in figure 10. It was observed that the drugabsorption profile of tacrolimus from within T-FNCs wassignificantly different from the profiles obtained for thebare drug administered via oral and intravenous routes. Asexpected, the retention time for tacrolimus was found to besignificantly enhanced for the nanoformulation compared tothe bare drug, implying its better bioavailability. For baretacrolimus administered via the oral route, an initial increasein Tmax value was recorded after 2 h, clearly indicating therapid absorption rate of the drug in SD rats wherein thetotal blood concentration reached a maximum of 0.312 mgduring this time period. Further on, a rapid decline in thedrug level was noted, reaching a negligibly low value of0.007 mg at 24 h. In contrast, the kinetics was much morecontrolled after the oral administration of T-FNCs, withthe drug levels peaking upto 0.216 mg at the sixth hourand showing a prolonged release pattern, with a slow andsteady decline upto 0.092 mg at 24 h. Great similaritieswere observed for the drug absorption profiles from T-FNCsrecorded for the two routes of administration. However, forthe bare drug given via the iv route, drug absorption wasmuch lower than that obtained for oral delivery. The moststriking observation was that the serum drug concentrationlevel at 24 h was high enough to sustain the drug releasefor longer time periods and enhance its therapeutic efficacywhen tacrolimus is administered as a nanoformulation. In
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Nanotechnology 23 (2012) 095102 G Praveen et al
Figure 11. In vitro release profile of VEGF conjugated fibrinnanotubes. The values represent the mean ± SD (n = 6) (p < 0.05,Anova).
view of the drug release deviation values for the tworoutes of administration, it is notable to mention that thecontrolled release kinetics of tacrolimus from T-FNCs forboth routes appear to be very similar, thereby allowing moreflexibility of administration. Overall, the results obtained forthe oral and iv administration of T-FNCs depict a distinctlydifferent pharmacological profile than the administration ofbare drug as reported earlier using nanoformulations [28]. Theadvantage of our system compared to other nanoformulationsis that fibrin being an endogenous material is well toleratedand does not account for any kind of toxic or immunologicreactions. This aspect can be taken to our advantagewhen designing the use of these fibrin nanoconstructs fordrug/growth factor delivery in tissue-engineered scaffolds.Since the release profile from the nanoformulation is slow,when incorporated into tissue-engineered scaffolds it canprovide a local delivery system with enhanced concentrationsfor prolonged time periods, thereby reducing the chances ofimmunorejection.
3.5. ELISA estimation for in vitro VEGF release
The precise estimation of free VEGF eluted to the culturemedium was detected by means of ELISA and it was observedthat there is no burst release during the initial period of in vitroincubation. The release profile of VEGF was much controlledupto day 4 releasing only upto 17% of the encapsulated VEGFfollowed by a rapid enhancement in release rates within day 7(figure 11). Within this period, it is measured that almost 84%of the encapsulated VEGF got released to the media. Thisparticular kinetics observed with V-FNCs make the systemideally compatible for initial in vitro trials by introducingangiogenic growth factors of desired concentrations [29, 30].
3.6. VEGF assisted cell proliferation and in vitro tubeformation of HUVEC
VEGF is known to induce mobilization and multifold increaseof endothelial cell proliferation which was all tested both inclinically relevant in vitro and in vivo systems [31, 32]. Fromour in vitro cell proliferation experiment, it is clearly evidentthat a substantial increase in total cell population was there
Figure 12. In vitro cell proliferation quantitative assay on HUVECcells.
on samples incubated with FNCS. A nearly two-fold increasewas observed in total cell count when compared to thegelatin coated TCPS (figure 12). For assessing the patternedalignment of HUVEC, we created zones of V-FNC richareas over the scaffold (figure 13(a)), whereas no chemotacticmigration was observed on a bare FNC embedded surface(figure 13(c)). The cell migration and extent of patternedalignment was found to be enhanced after seven days ofculture incubation as more CD 31 expressing functionalendothelial cells were found to be aligned at the angiogeniczone (figure 13(f)). Since fibrin is an important naturalextracellular matrix component that is actively involved inwound healing and angiogenesis, it could also be suggestedthat the matrix assisted cell migration and tube formationis promoted by the cumulative action of systemic deliveryof VEGF and the subsequent induction of fibrin degradationproducts released within the system.
4. Conclusions
In this study we have exploited the possibility of synthesizingthe fibrin nanoconstructs of desired dimensions through asurfactant-free water-in-oil emulsification–diffusion process.Without distorting the molecular integrity of fibrin, conju-gation of the immunosuppressive drug, namely, tacrolimus,with fibrin nanoconstructs was performed. The feasibility ofusing FNCs loaded with desired growth factors as an idealmatrix protein component to induce in vitro vascularizationwas also exploited in this study. Drug loaded T-FNCs thusobtained had an encapsulation efficiency of 66% and alsoproved to have no direct toxicological effects when testedin vitro on hMSCs. The enhanced therapeutic efficiencyof the immunosuppressive drug when encapsulated withinFNCs and delivered to the target sites is of considerableinterest, particularly after transplantation surgeries. Thus, ourstudy findings highlight the potential advantage of usingfibrin based nanoconstructs for biomedical applications, asan ideal autologous biocomponent that prevents the riskof initial phase immunorejection. Moreover, the controlleddelivery of VEGF from the FNC conjugate induces rapidmigration of the endothelial cells toward the VEGF gradientand tube formation, thereby promoting in vitro vascularization
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Nanotechnology 23 (2012) 095102 G Praveen et al
Figure 13. (a) SEM of agarose scaffold showing the multi-tiered architecture and V-FNC rich zone (b) magnified view of the V-FNC richzone (indicated by arrows). (c) SEM of HUVEC fixed after 24 h incubation exhibiting no chemotaxis in response to the bare FNCs.(d) HUVEC cells exhibiting initial chemotactic movement in response to V-NCs. ((e) and (f)) HUVEC cells after seven-day incubation inresponse to bare FNCs and V-FNCS. Cells stained red using Texas red-labeled anti-CD31 and nuclei stained with DAPI. The patternedalignment of cells is indicated by arrows.
of 3D scaffolds. Altogether, these novel fibrin nanoconstructsdeveloped using a green chemistry approach, proved to beexcellent biodegradable nanocarriers for oral as well asparenteral administrations, with remarkable potential also fordelivering specific drugs/growth factors in tissue engineeringscaffolds.
Acknowledgments
The authors are highly indebted to the Department of Scienceand Technology, Government of India for the financialassistance under the Nanoscience and NanotechnologyInitiative program monitored by Professor C N R Rao. Specialthanks are given to Professor A K K Unni for his help withthe animal testing, Mrs Dhanya Narayanan, Mr Sajin P Ravi,and Mr C M Girish for their assistance in HPLC, SEM,and AFM analysis respectively. The authors are also gratefulto the Amrita Institute of Medical Sciences for providinginfrastructural support for this project.
DisclosuresThe authors declare that no competing financial interests
exist.
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