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Jun 2, 2016 - and Biological Evaluation of Poly(Glutamic ... Keywords: poly(amino acid)s; amphiphilic copolymers; polymer particles; polymersomes;.
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Preparation, Characterization, and Biological Evaluation of Poly(Glutamic Acid)-b-Polyphenylalanine Polymersomes Evgenia Vlakh 1,2 , Anastasiia Ananyan 2 , Natalia Zashikhina 2 , Anastasiia Hubina 1 , Aleksander Pogodaev 1 , Mariia Volokitina 1,2 , Vladimir Sharoyko 1 and Tatiana Tennikova 1,2, * 1

2

*

Institute of Chemistry, Saint-Petersburg State University, Universitesky pr. 26, 198504 St. Petersburg, Russia; [email protected] (E.V.); [email protected] (A.H.); [email protected] (A.P.); [email protected] (M.V.); [email protected] (V.S.) Institute of Macromolecular Compounds, Russian Academy of Sciences, Bolshoy pr. 31, 199004 St. Petersburg, Russia; [email protected] (A.A.); [email protected] (N.Z.) Correspondence: [email protected]; Tel.: +7-812-323-0461

Academic Editor: Sebastien Lecommandoux Received: 18 March 2016; Accepted: 25 May 2016; Published: 2 June 2016

Abstract: Different types of amphiphilic macromolecular structures have been developed within recent decades to prepare the polymer particles considered as drug delivery systems. In the present research the series of amphiphilic block-copolymers containing poly(glutamatic acid) as hydrophilic, and polyphenylalanine as hydrophobic blocks was synthesized and characterized. Molecular weights for homo- and copolymers were determined by gel-permeation chromatography (GPC) and amino acid analysis, respectively. The copolymers obtained were applied for preparation of polymer particles. The specific morphology of prepared polymerosomes was proved using transmission electron microscopy (TEM). The influence on particle size of polymer concentration and pH used for self-assembly, as well as on the length of hydrophobic and hydrophilic blocks of applied copolymers, was studied by dynamic light scattering (DLS). Depending on different experimental conditions, the formation of nanoparticles with sizes from 60 to 350 nm was observed. The surface of polymersomes was modified with model protein (enzyme). No loss in biocatalytic activity was detected. Additionally, the process of encapsulation of model dyes was developed and the possibility of intracellular delivery of the dye-loaded nanoparticles was proved. Thus, the nanoparticles discussed can be considered for the creation of modern drug delivery systems. Keywords: poly(amino acid)s; amphiphilic copolymers; polymer particles; polymersomes; encapsulation; biodegradation; cell uptake

1. Introduction The development of modern drug delivery systems to solve the problem of directed transport at cellular and sub-cellular scales reduces both the probability of loaded drug degradation and its high toxicity in the body [1]. The variety of polymer materials opens up wide possibilities to create such systems of tunable morphology appropriate to the size of the organism. Different types of carriers have been constructed for drug delivery applications, including polymer conjugates, nanogels, along with nano- and microparticles of different morphologies [2–4]. Recently, elevated attention has been paid to the polymer nanovesicles of a core-shell structure with double layer liposome-like membranes [5–8]. The structure of such a polymer shell has much in common with that of a cell membrane, which can enhance the cell permeability for developed nanoparticles. These nanocarriers, composed of the amphiphilic block-copolymers, are known under the name polymersomes and, compared to liposomes, demonstrate higher membrane stability (mainly due to the membrane thickness, which can exceed Polymers 2016, 8, 212; doi:10.3390/polym8060212

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9 nm) and tunable membrane properties [9–11]. The thicker membrane prolongs the stability of the nanocarrier in a blood flow and enhances the therapeutic efficacy of the loaded drug [11]. Furthermore, the amphiphilic membrane of polymersomes provides the advantage to integrate both hydrophilic and hydrophobic compounds, such as drugs, genes, imaging agents, etc. [1,4,7]. The unique controllable structure of polymersomes allows the construction of nanovesicles with stimuli response depending on temperature, pH value, or ionic strength change, which can disassemble and release the encapsulated drug [12,13]. Different types of amphiphilic structures have been developed in recent decades, including dendritic compounds, graft, di-, and triblock copolymers. PEG is widely used as the most common neutral hydrophilic constituent of di- and triblock copolymers, or as a fragment of polymer brushes due to its low toxicity and high stability [14,15]. A lot of polymers are applied as building blocks for polymersomes, namely, polypropylene oxide, polylactic acid, polybutadiene [16], etc. Despite the wide diversity of building blocks for the structures discussed, the polypeptides represent one of the most attractive classes of polymers for polymersome formation. These polymers combine the advantages of synthetic polymers with peptides’ ability to form secondary structure, biocompatibility, functionality, and biodegradability due to their units produced as a result of degradation and representing the metabolic substances [3,17–19]. Moreover, it is well known that polypeptides can change their conformation under the variation of temperature or pH that can be used for construction of stimuli-responsive polymersomes [20]. The variety of functional groups located on the polymersome surface provides the possibility of nanocarrier modification with bioligands to create the systems for controlled drug delivery. There are two main methods for polypeptide synthesis, e.g., the step-wise in-solution or solid-phase condensation of activated amino acids, and the ring-opening polymerization (ROP) of α-amino acid N-carboxyanhydrides (NCA) [18,21,22]. The first way is suitable for synthesis of heteropeptides with well-defined sequences and lengths up to 100 residues. However, this approach is not appropriate for direct preparation of large polypeptides, especially homopolypeptides, because of its laborious, time-consuming, economic inefficiency. The most expedient way for synthesis of long chain homopolypeptides, random polypeptides, graft-, or block polypeptides, is the ring-opening polymerization (ROP) of N-carboxyanhydrides of α-amino acids. This approach represents a reliable method to obtain polypeptides of controllable molecular weight and low polydispersity index with no racemization [23]. A number of block copolypeptides, which are able to self-assemble into micelles or vesicles, was described in the literature. For example, Kim et al. [24] described the synthesis of poly(L-glutamic acid)-b-poly(L-phenylalanine) copolymers with small hydrophobic blocks consisted of 2–7 phenylalanine units for the preparation of particles. Holowka et al. [25] reported the development of charged polypeptide vesicles based on poly(L-lysine)60 -b-poly(L-leucine)20 , poly(L-glutamic acid)60 -b-poly(L-leucine)20 or poly(L-argenine)60 -b-poly(L-leucine)20 polypeptides appropriate for drug encapsulation. However, no consideration of the effect of block length or conditions of self-assembly on particle properties was given in these papers [24,25]. In Huang et al. [26] the influence of block copolymer length on the formation of poly(L-lysine)-b-poly(L-tyrosine) particles was discussed. The investigation of biocompatibility of hydrogels or particles based on block copolypeptides containing L-lysine, L-glutamic acid, or L-argenine for the building of hydrophilic blocks, and L-leucine for the construction of hydrophobic ones, revealed the absence of cytotoxicity [27–29]. Nevertheless, not too much attention was paid to the cytotoxicity of high molecular weight block copolypeptides, which are more prospective considering their higher stability and prolonged release in comparison to the low molecular weight analogues. In the current research the series of poly(L-glutamic acid)-block-poly(L-phenylalanine) (PGlu-bPPhe) samples with polypeptide blocks containing up to 117 L-glutamic acid and 165 L-phenylalanine residues was obtained and used for the preparation of nanoparticles. The peculiarities of morphology of constructed nanoparticles were investigated. The biodegradation of PGlu-b-PPhe nanoparticles was evaluated using in vitro degradation in a model enzymatic system and human blood plasma. The cytotoxicity of polymersomes obtained was tested using two cell lines, namely, HEK and Caco-2.

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Additionally, the process of encapsulation of model dyes was developed and the possibility of intracellular delivery of the dye-loaded nanoparticles was proved. 2. Materials and Methods 2.1. Materials γ-benzyl-L-glutamate, L-phenylalanine, triphosgene, α-pinene, n-hexylamine (HEXA), trifluoromethanesulfonic acid (TFMSA), trifluoroacetic acid (TFA), N-hydroxysuccinimide (NHS), 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (CDI), bromophenol blue, rhodamine 6g, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT), and other reagents were purchased from Sigma-Aldrich (Darmstadt, Germany) and used as received. 1,4-dioxane, n-hexane, N,Ndimethylformamide (DMF), dimethyl sulfoxide (DMSO), ethyl acetate, methanol, and other solvents were purchased from Vecton Ltd. (St. Petersburg, Russia) and distilled before application. All salts used for buffers preparation were also purchased from Vecton Ltd. and were of ACS reagent grade. The buffer solutions were prepared by dissolving salts in distilled water and additionally purified by filtration through a 0.45-µm membrane microfilter Milex, Millipore Inc. (Hietzinger, Austria). Vivaspin concentrators (30,000) used for ultrafiltration were products of Sartorius (Goettingen, Germany). The Spectra/Pore® (MWCO: 1000) dialysis bags were purchased from Spectra (Rancho Dominguez, CA, USA). Human embryonic kidney cells (HEK 293) and human epithelial colorectal adenocarcinoma cells (Caco-2) were obtained from BioloT (St. Petersburg, Russia) and were grown in Dulbecco’s Modified Eagle’s Medium (BioloT, St. Petersburg, Russia) containing 10% (v/v) heat-inactivated fetal bovine serum (FBS, HyClone Laboratories, Logan, UT, USA), 1% L-glutamine, 1% sodium pyruvate, 50 U/mL penicillin, and 50 µg/mL streptomycin (BioloT). 2.2. Instrumentation The structure and purity level of synthesized NCAs were confirmed by 1 H NMR. Spectra were recorded at 298 K using a Bruker 400 MHz Avance instrument (Karlsruhe, Germany) and CDCl3 . Gel permeation chromatography (GPC) measurements were performed on Shimadzu LC-20 Prominence system with refractometric RID 10-A detector (Kyoto, Japan) using 7.8 mm ˆ 300 mm Styragel Column, HMW6E, 15–20 µm bead size (Waters, Milford, MS, USA). The analysis was carried out at 60 ˝ C using DMF with 0.1 M LiBr as eluent. The mobile phase flow rate was 0.3 mL/min. Molecular weights and molecular weight distributions for γ-Glu(Bzl) homopolymers were calculated using poly(methyl methacrylate)standards with Mw range from 17,000 to 250,000 g/mol and polydispersity lower than 1.14. The calculations were carried out using GPC LC Solutions software (Shimadzu, Kyoto, Japan). The contribution of hydrophobic block was determined using chromatographic amino acid analysis after total hydrolysis of the samples. The hydrolyzate was analyzed by reversed-phase (RP) high-performance liquid chromatography (HPLC) using a Shimadzu system with UV-detection (Kyoto, Japan) equipped with 2 mm ˆ 150 mm Luna C18 column, packed with 5 µm particles. The isocratic elution mode was applied and 0.1% acetonitrile/HCOOH in a ratio 5/95 wt % was used as eluent. The mobile phase flow rate was equal to 0.3 mL/min. To study the influence of polymer composition, as well as different parameters used in the preparation step, the DLS method was used for particle size characterization. DLS measurements were performed on a SZ100 (Horiba JobinYvon, Kyoto, Japan) laser particle analyzer at a scattering angle of 90˝ at 25 ˝ C. The range of concentrations of nanoparticles in Na-borate buffer solution, pH 8.7, was 0.5 and 0.25 mg/mL. The morphological peculiarities were investigated using transmission electronic microscopy (TEM) using a Jeol JEM-2100 (Tokyo, Japan) microscope operated at an acceleration voltage of 160 kV. Before analysis, a few drops of sample were placed onto a copper grid covered with carbon for 30 s. The dried grid was stained negatively with 2% (w/v) uranyl acetate solution for 30 s and used for measurements after 24 h.

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2.3. Synthesis and Particles Preparation NCAs of γ-Glu-(Bzl) and Phe were synthesized as described elsewhere [30]. Dioxane was used as a solvent, and acquired NCA was purified by recrystallization from ethyl acetate/n-hexane. Yields: NCA of γ-Glu-(Bzl)—75%, NCA of Phe—70%. Structure and purity level of synthesized NCAs were confirmed by 1 H NMR. 1 H NMR: NCA of γ -Glu-(Bzl)—2.05–2.39 (m, 2 H), 2.63 (t, 2 H), 4.39 (t, 1 H), 5.17 (s, 2 H), 6.40 (br. s., 1 H), 7.39 (m, 5 H), NCA of Phe: 2.94–3.35 (m, 2H), 4.55 (m, 1H), 5.60 (s, 1H), 7.19–7.41 (m, 5H). The ring opening polymerization (ROP) of γ-Glu-(Bzl) NCA was carried out using HEXA as initiator at 4% of monomer in 1,4-dioxane. The polymerization was carried out for 24 h at 30 ˝ C. Then the polymer was precipitated, washed with diethyl ether three times, and dried. Then, p-Glu-(Bzl) copolymerization with Phe-NCA was carried out in DMF for 48 h at 30 ˝ C to obtain a series of P-γ-Glu(Bzl)-b-PPhe with various P-Glu/PPhe balance. The Bzl-protective group of P-γ-Glu(Bzl)-b-PPhe was removed by TFMSA/TFA mixture in a ratio 1/10 at 22 ˝ C. The samples were totally hydrolyzed and the products of hydrolysis were investigated by RP-HPLC as described in Section 2.2. The hydrolysis of 1 mg of a sample was carried out in 2 mL of 6 M HCl with 0.0001% phenol in vacuum-sealed ampoule for four days. The solvent was evaporated several times with water to eliminate HCl and to reach finally the neutral pH value. After deprotection, the product was dispersed in DMSO, put into a dialysis membrane bag MWCO 1000, and dialyzed against Na-borate buffer solution, pH 8.6, for one day. After two days of freeze drying, PGlu-b-PPhe was collected. Polymer nanoparticles were prepared by phase inversion during dialysis, followed by freeze drying and final dispersing for 2 h under sonication at necessary concentration (0.25–1.00 mg/mL) in the corresponding buffer (Na-phosphate or Na-borate buffer solutions, pH 7.4–10.5). 2.4. Biodegradation study To study the biodegradation process of PGlu-b-PPhe nanoparticles, the accumulation of free amino acids in a medium was controlled. 0.02 M Na-phosphate buffer, pH 7.4, containing hydrolase papain, were used as model physiological conditions. For that, 50 µg of enzyme was introduced into suspension to reach the volume of 1 mL. In parallel, 1 mg of nanoparticles in 100 µL of 0.02 M Na-phosphate buffer, pH 7.4, was added to 900 µL of human blood plasma. All reactions of nanoparticle degradation were carried out for three months at 37˝ C and monitored by offline cation exchange HPLC of the reaction products (Glu and Phe amino acids). For this purpose, the commercially available methacrylate-based ultra-short monolithic column, namely, CIMSO3 disk (3 mm ˆ 12 mm i.d.) (BIA Separations, Ajdovscina, Slovenia) was applied. UV detection was performed at 210 nm. The data was acquired and processed with LS Solution software (Shimadzu, Kyoto, Japan). 0.02 M aqueous Klark-Labbs buffer, pH 2.0, (eluent A), 0.02 M Na-phosphate buffer, pH 7.0, (eluent B) and 0.0125 M Na-borate buffer, pH 10.0 (eluent C), were used as the mobile phases for HPLC of Glu and Phe. The separation was carried out using follow protocol: 0–0.5 min—eluent A, 0.5–7 min—eluent B, 7–10 min—eluent C at a flow rate of 0.5 mL/min. 2.5. Surface Modification The modification of the surface of PGlu62 -b-PPhe82 nanoparticles with the model protein, namely, ribonuclease A, was carried out after preliminary activation of carboxylic groups. The nanoparticles were prepared in 0.01 M Na-borate solution, pH 8.4, and then dialyzed using a 10 kDa dialysis membrane against 0.01 M MES buffer, pH 6.0. 4 mL of suspension with a concentration of 0.5 mg/mL was mixed with a two-fold excess of NHS and CDI required to activate 20% of Glu units. The activation was carried out at 4 ˝ C for 30 min. Then, 2 mg of ribonuclease was added to the suspension of activated nanoparticles and left under stirring conditions for 30 min at 20 ˝ C. The excess of the enzyme was removed via dialysis using MWCO 30,000 membrane. The amount of immobilized enzyme was evaluated using the Lowry method [31]. The activity of bound and free ribonuclease was determined using low molecular weight-specific substrate 2,3-cytidine cyclophosphate and methodology published elsewhere [32].

ribonuclease was determined using low molecular weight-specific substrate 2,3-cytidine cyclophosphate and methodology published elsewhere [32]. 2.6. Encapsulation of Dyes Polymers 2016, 8, 212

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For encapsulation of bromophenol blue, 4.0 mL solutions of dye with concentrations from 0.05 to 2.50 mg/mL in 0.01 M Na-borate buffer, pH 9.5, were prepared, then added to 4.0 mg of block 2.6. Encapsulation of Dyes copolymer, mixed, and sonicated for 4 h. The free dye was separated from particles by gel-filtration using a For Sephadex G-100 column of 0.8 (i.d.) × 30.0 mm2. of The part of concentrations encapsulated from dye was encapsulation gel of bromophenol blue, 4.0 mL solutions dye with calculated as amg/mL difference totalbuffer, and free dye amounts, the latter was determined 0.05 to 2.50 in 0.01between M Na-borate pH 9.5, were prepared, then added to 4.0 mg of blockusing copolymer, mixed, andmeasured sonicated for 4 h. nm The and free dye was separated from particles by gel-filtration spectrophotometric data at 590 corresponding calibration curve. 2 . The part of encapsulated dye was using a Sephadex G-100 gel column of 0.8 (i.d.) ˆ 30.0 mm Rhodamin loading was performed as follows: 0.5 mg of rhodamin 6g dissolved in 0.4 mL calculated difference betweenand total3.6 and thebuffer, latter was determined using and DMSO, 1.0 mgasofa block copolymer mLfree of dye 0.01 amounts, Na-borate pH 10.5, were mixed spectrophotometric measured at 590 nmDMSO and corresponding calibration curve. through a 30 kDa sonicated for 6 h. Thedata excess of dye and were removed via dialysis Rhodamin loading was performed as follows: 0.5 mg of rhodamin 6g dissolved in 0.4 mL DMSO, MWCO membrane until no fluorescence was observed in a filtered solution. 1.0 mg of block copolymer and 3.6 mL of 0.01 Na-borate buffer, pH 10.5, were mixed and sonicated for 6 h. The excess of dye and DMSO were removed via dialysis through a 30 kDa MWCO membrane 2.7. Cell Experiments until no fluorescence was observed in a filtered solution.

The cells were routinely cultured at 37 °C in a humidified atmosphere containing air and 5% CO2. Cytotoxicity of colloid solutions of polymer nanoparticles with concentrations 0.05–0.5 mg/mL ˝ C in a humidified atmosphere containing air and 5% CO . The cells were cultured at 37 test 2 was evaluated with routinely a standard MTT as described elsewhere [33]. The incubation of Cytotoxicity of colloid solutions of polymer nanoparticles with concentrations 0.05–0.5 mg/mL was nanoparticles with cells was performed for 48 h. The values measured at 540 nm were subtracted for evaluatedcorrection with a standard described elsewhere incubation of nanoparticles background at 690MTT nm,test andasthe data was plotted[33]. as aThe percent of control samples using with cells was performed for 48 h. The values measured at 540 nm were subtracted for background Microsoft Excel software (Microsoft Corp., Redmond, WA, USA). correction at 690 nm, and the data was plotted as a percent of control samples using Microsoft Excel To monitor if the particles penetrate the cells, 200 μL of cell culture medium containing Caco-2 software (Microsoft Corp., Redmond, WA, USA). cells were glass chamber slides with CC2 treatment) cultured for 24 h. To seeded monitor on if the particles penetrate the(LabTec-II cells, 200 µL of cell culture mediumand containing Caco-2 Then, medium fresh one containing cellsthe wereincubation seeded on glass chamberwas slideschanged (LabTec-IIwith with CC2 treatment) and culturedrhodamine-loaded for 24 h. Then, polymersomes and the mixtures were incubated for 4 h. After that, the cells were washed the incubation medium was changed with fresh one containing rhodamine-loaded polymersomes andthree times warm PBS solution fluorescence microscope (Olympus thewith mixtures were incubated forand 4 h. observed After that, using the cellsa were washed three times with warm PBSIX50, solution and observed using aequipped fluorescence microscope (Olympus IX50, Olympus Corp.,the Tokyo, Japan) Olympus Corp., Tokyo, Japan) with a SC30 Olympus camera to capture images of cells equipped with a SC30 Olympus camera to capture the images of cells (excitation filter: BP 530–550, (excitation filter: BP 530–550, barrier filter: BA590). The images were acquired at 20× optical zoom. 2.7. Cell Experiments

barrier filter: BA590). The images were acquired at 20ˆ optical zoom.

3. Results and Discussion

3. Results and Discussion

3.1. Synthesis of Poly(Amino Acid) 3.1. Synthesis of Poly(Amino Acid)Block-Copolymers Block-Copolymers general schemeofofpolymer polymer synthesis synthesis isispresented in Figure 1. As1.aAs firsta step, synthesis of TheThe general scheme presented in Figure first the step, the synthesis several homopolymers of γ-Glu(Bzl) was carried out using the method of ring-opening polymerization of several homopolymers of γ-Glu(Bzl) was carried out using the method of ring-opening of the corresponding NCA. polymerization of the corresponding NCA.

Figure 1. 1.Scheme synthesis of amphiphilic PGlu-b-PPhe copolymers for preparation of Figure Scheme ofofsynthesis of amphiphilic PGlu-b-PPhe copolymers for preparation of polymersomes. polymersomes.

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The Thecharacteristics characteristicsofofsynthesized synthesizedP-γ-Glu(Bzl)s P-γ-Glu(Bzl)sare arecollected collectedininTable Table1.1.As Asaasecond secondstep, step,the the P-γ-Glu(Bzl) was used as macroinitiator for copolymerization of the hydrophobic block of Phe-NCA. P-γ-Glu(Bzl) was used as macroinitiator for copolymerization of the hydrophobic block of The formation of formation amphiphilic copolymers achieved after deprotection the Bzl-protective Phe-NCA. The of block amphiphilic blockwas copolymers was achieved afterofdeprotection of the group. Thus, four samples differing with the length of the hydrophilic and hydrophobic blocks were Bzl-protective group. Thus, four samples differing with the length of the hydrophilic and prepared (Table 2). hydrophobic blocks were prepared (Table 2). Table 1. Molecular mass characteristics of P-γ-Glu(Bzl). Table 1.Molecular mass characteristics of P-γ-Glu(Bzl). Sample Sample M nMn

BG1 BG1 BG2 BG2

M wMw M w n n nn M/M w/M

13,800 16,600 16,600 1.21.2 13,800 25,800 25,800 36,100 36,100 1.41.4

6262 117 117

Table Table2.2.Molecular Molecularmass masscharacteristics characteristicsofofsynthesized synthesizedamphiphilic amphiphilicPGlu-b-PPhe PGlu-b-PPhecopolymers. copolymers.

Sample MM n (PGlu) Sample n (PGlu) GP1 GP1 GP2 GP2 GP3 GP3 GP4 GP4

8,000 8,000 8,000 8,000 15,100 15,100 15,100 15,100

nn

62 62 62 62 117 117 117 117

M M nn (PPhe) (PPhe) 4,600 4,600 12,100 12,100 11,900 11,900 24,200 24,200

content,%% m m Hydrophilic Hydrophilicblock block content, 31 64 31 64 82 40 82 40 81 56 81 56 165 38 165 38

3.2. 3.2.Characterization CharacterizationofofParticles Particles To Toprepare preparethe theparticles, particles,“phase “phaseinversion” inversion”method, method,which whichwas wasapproved approvedasasthe themost mostsuitable suitable totoobtain obtainthe thenanoobjects nanoobjectswith witha apolymersome polymersomestructure structure[9], [9],was wasapplied. applied.ItItisisknown knownthat thatseveral several factors factorsinfluence influencethe theself-assembling self-assemblingbehavior behaviorofofamphiphilic amphiphilicpolymers. polymers.Particularly, Particularly,the thelength lengthofof hydrophobic/hydrophilic pH, pH, and concentration are of great the characteristics hydrophobic/hydrophilicblocks, blocks, and concentration areimportance of great for importance for the ofcharacteristics the formed particles. of the formed particles. First Firstofofall, all,the theeffect effectofofthe theblock blocklength lengthon onthe theparticle particlehydrodynamic hydrodynamicdiameter diameterwas wasevaluated evaluated (Figure Thecomparison comparisonof of polymers containing the hydrophilic thelength, same namely, length, (Figure2). 2). The polymers containing the hydrophilic blockblock of the of same namely, GP1GP2 with GP2 andwith GP3GP4, withallowed GP4, allowed the conclusion that the increase the length GP1 with and GP3 the conclusion that the increase of theoflength of the ofhydrophobic the hydrophobic the formation of larger particles. in agreement blockblock led toled thetoformation of larger size size particles. This This resultresult is inisagreement with with published self-assembly otherkinds kindsof of amphiphilic amphiphilic block-copolymers, published datadata on on self-assembly of ofother block-copolymers, for forinstance, instance, poly(ethylene [34][34] and poly(ethylene glycol)-b-polystyrene [35]. When poly(ethyleneglycol)-b-poly(ε-caprolactone) glycol)-b-poly(ε-caprolactone) and poly(ethylene glycol)-b-polystyrene [35]. the hydrophilic block of block PGlu-b-PPhe was increased, but the hydrophobic block remained When the hydrophilic of PGlu-b-PPhe was increased, but the hydrophobic block constant remained (samples and GP3) a minor decrease a particleinhydrodynamic diameter was observed. constantGP2 (samples GP2 only and GP3) only a minorindecrease a particle hydrodynamic diameter was This effect can related highertorepulsion charged-like polymer chains with chains the growth observed. Thisbeeffect cantobethe related the higherofrepulsion of charged-like polymer with of the Glu-block and, length as a result, in a smaller in sized particles. growth oflength Glu-block and,the as aself-assembly result, the self-assembly a smaller sized particles.

Figure 2. Effect of block length on particle size (particle concentration is 0.5 mg/mL, pH 8.4). Figure 2. Effect of block length on particle size (particle concentration is 0.5 mg/mL, pH 8.4).

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The effect of pH on particle hydrodynamic diameter was examined using the series of 0.01 M 7 of 14Since pH 7.4, 8.4, 9.5, and 10.5 and two polymer samples, namely GP1 and GP3. 7 of 14 the tendency for both polymers was the same, the data for GP1 particles are presented as an example in FigureThe 3. effect It is obvious the hydrodynamic hydrodynamic diameter nanoparticles slightly decreased of pH on that diameter was examined using series 0.01 M in The particle diameter wasof examined using the the series of of buffer solutions 8.4, 9.5, and 10.5 and two polymer samples, namely GP1conditions and GP3. Since alkaline media. Thiswith canpH be 7.4, attributed to the better PGlu solubility under alkaline because the tendency for both polymers was the same, the data for GP1 particles are presented as an example for formation GP1 particles presented example of of the higher ionization degree, which is favoreddata to the ofare random coil conformation in Figure It is obvious that the hydrodynamic diameter of nanoparticles slightly decreased in 3. It is obvious that the hydrodynamic diameter nanoparticles slightly decreased polypeptides [36]. Polymers 2016, 8,with 212 buffer solutions Polymers 2016, 8, 212

alkaline media. This This can can be be attributed attributed to to the the better better PGlu PGlu solubility solubility under under alkaline alkaline conditions conditions because of the higher ionization degree, which is favored to the formation of random coil conformation conformation of polypeptides [36].

Figure 3. Dependence of particle size on pH (polymer sample GP1, concentration is 0.5 mg/mL). Figure 3. 3. Dependence on pH pH (polymer (polymer sample sample GP1, GP1, concentration concentration is is 0.5 0.5 mg/mL). mg/mL). Figure Dependence of of particle particle size size on

The effect of block copolymer concentration on particle hydrodynamic diameter was The within effect ofthe block copolymer concentration0.25, on particle hydrodynamic was investigated following concentrations: 0.50, and 1.00 mg/mLdiameter for all polymer The effect of block copolymer concentration on particle hydrodynamic diameter was investigated investigated within the following concentrations: 0.25, 0.50, and 1.00 mg/mL for all polymer samples. defined concentrations: values of nanoparticle hydrodynamic did not show significant withinThe the following 0.25, 0.50, and 1.00 mg/mL fordiameter all polymer samples. The defined samples. The defined values of nanoparticle hydrodynamic diameter did not show significant change with the growth hydrodynamic of concentration. As andid example, DLS data for the prepared values of nanoparticle diameter not showthe significant change withsamples the growth of change with the growth of concentration. As an example, the DLS data for the samples prepared fromconcentration. GP3 copolymer are shown in Figure 4. Additionally, the influence of NaCl concentration As an example, the DLS data for the samples prepared from GP3 copolymer are shown was from GP3 copolymer are shown in Figure 4. Additionally, the influence of NaCl concentration was in Figure Additionally, the influence of NaCl concentration studied. No change inNaCl particle studied. No 4.change in particle size was observed in thewas range of 0.3%–2.0% in size 0.01 M studied. No change in particle size was observed in the range of 0.3%–2.0% NaCl in 0.01 M was observed in the range of 0.3%–2.0% NaCl in 0.01 M Na-phosphate buffer (PBS), pH 7.4 (Figure 5). Na-phosphate buffer (PBS), pH 7.4 (Figure 5). Na-phosphate buffer (PBS), pH 7.4 (Figure 5).

Figure 4. Dependence of particle size on polymer concentration (polymer sample GP3, pH 8.4). Figure 4. Dependence particlesize sizeon onpolymer polymer concentration sample GP3, pH 8.4). Figure 4. Dependence of of particle concentration(polymer (polymer sample GP3, pH 8.4).

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Figure 5. Effect of salt concentration on mean size of nanoparticles (polymer sample GP3, pH 7.4, Figure 5. Effect of salt concentrationon onmean mean size size of sample GP3, pH 7.4, Figure 5. Effect of salt concentration of nanoparticles nanoparticles(polymer (polymer sample GP3, pH 7.4, particle concentration 0.5 mg/mL). particle concentration 0.5 mg/mL). particle concentration 0.5 mg/mL).

One of the most important characteristics of colloidal systems is ξ-potential, ξ-potential, which reflects their One of the most important characteristics ofiscolloidal systems is ξ-potential, which reflects their stability regarding suspension aggregation. ItIt is known that that colloids colloids are considered to be stable known stability suspension It is known that considered to befor stable whenregarding the ξ-potential is than than +30 mVcolloids [37]. Theare values of ξ-potential ξ-potential is lower loweraggregation. than ´30 −30 and higher ξ-potential whenparticles the ξ-potential is lower diameter than −30up and higher thanwere +30 around mV [37]. The values of ξ-potential with to 350 around ´70–50 mV at mV pH 7.4, which particles withhydrodynamic hydrodynamic diameter up to nm 350 were nm −70–50 at pH 7.4,allowed which for particles with hydrodynamic up tothe nm were around of −70–50 mV at particles pH 7.4,the which assumption of their high To stability. examine stability ofthe PGlu-b-PPhe particles on aggregation, allowed assumption of stability. theirdiameter high To350 examine stability PGlu-b-PPhe on experiments monitoring the particle size for two weeks at physiological conditions (PBS buffer, pH 7.4, allowed assumption of their high stability. examine thetwo stability ofphysiological PGlu-b-PPheconditions particles on aggregation, the experiments monitoring theTo particle size for weeks at ˝ C) were carried out (Figure 6). According to DLS data (Figure S1 of Supplementary Material), 37 (PBS buffer, pH 7.4, 37 °C) were carried (Figure According to at DLS data (Figureconditions S1 of aggregation, the experiments monitoring theout particle size6).for two weeks physiological no other peaks, apart from a single one corresponding to initial-sized particles, were observed within Supplementary Material), no other peaks, apart from a single one corresponding to initial-sized (PBS buffer, pH 7.4, 37 °C) were carried out (Figure 6). According to DLS data (Figure S1 of the tested period. Thus, the nanoobjects can beThus, ascorresponding a stablenanoobjects colloid system particles, were observed within tested period. the one developed canwith be Supplementary Material), nodeveloped other the peaks, apart from acharacterized single to initial-sized no evident tendency to aggregation. characterized as a stable colloid system with no evident tendency to aggregation. particles, were observed within the tested period. Thus, the developed nanoobjects can be

characterized as a stable colloid system with no evident tendency to aggregation.

Figure 6. 6. Storage Storage stability stability of of the the GP3 GP3 colloid colloid system system at atphysiological physiologicalconditions. conditions. Conditions: Conditions: PBS PBS Figure buffer of pH 7.4; temperature 37 °C; concentration 0.5 mg/mL. buffer of pH 7.4; temperature 37 ˝ C; concentration 0.5 mg/mL.

Figure 6. Storage of the GP3block colloid system at physiological conditions. It is known stability that amphiphilic copolymers can self-assemble into aConditions: wide rangePBS of It is known that amphiphilic block copolymers can self-assemble into a wide range of morphologies [6];temperature for example, cylindrical, spherical micelles, or polymeric vesicles. The formed buffer of pH 7.4; 37 °C; concentration 0.5 mg/mL. morphologies [6]; for example, cylindrical, spherical micelles, or polymeric vesicles. The formed particles can be definitely identified as polymer vesicles, or polymersomes (Figure 7) possessing a particles can be definitely identified as polymer vesicles, or polymersomes (Figure 7) possessing a polymer membrane (double dark block circle) copolymers and aqueouscan coreself-assemble (light interior). the of It is known that amphiphilic intoConsidering a wide range polymer membrane (double dark circle) and aqueous core (light interior). Considering the hydrophobic hydrophobic block presented by polyphenylalanine, the observed structure can be formed due to morphologies [6]; for example, cylindrical, spherical micelles, or polymeric vesicles. The formed block presented by polyphenylalanine, the observed structure can be formed due to π–π interactions π–π can interactions between aromaticasfragments of hydrophobic chains. Despite the copolymers particles be definitely identified polymer vesicles, or polymersomes (Figure possessing a between aromatic fragments of hydrophobic chains. Despite the copolymers applied to the 7) preparation applied to the preparation of particles differing with the length of the macromolecules and the polymer membrane and aqueousand core interior). Considering of particles differing(double with the dark length circle) of the macromolecules the (light length of hydrophobic block, the the length of hydrophobic block, the morphology for all samples prepared in the pH range 7.4–10.5 and hydrophobic block presented by polyphenylalanine, the observed structure can be formed due to concentration up to 1.0 mg/mL remained the same.

π–π interactions between aromatic fragments of hydrophobic chains. Despite the copolymers applied to the preparation of particles differing with the length of the macromolecules and the length of hydrophobic block, the morphology for all samples prepared in the pH range 7.4–10.5 and concentration up to 1.0 mg/mL remained the same.

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Polymersmorphology 2016, 8, 212 for all samples prepared in the pH range 7.4–10.5 and concentration up to 1.0 mg/mL 9 of 14

remained the same.

Figure 7. TEM images of polymer nanoparticles obtained (pH 8.4, concentration 0.5 mg/mL).

Figure(A) 7. Polymersomes TEM images (GP2); of polymer nanoparticles obtained and (B) single polymersome (GP3). (pH 8.4, concentration 0.5 mg/mL). (A) Polymersomes (GP2); and (B) single polymersome (GP3). 3.3. Biodegradation

3.3. Biodegradation The degradation of poly(amino acids) in biological environments occurs only when catalyzed by enzymes. The rateof of poly(amino degradation depends on biological the specificity of involved enzymes. complication The degradation acids) in environments occurs The only when catalyzed of enzymatic degradation is connected with the time-scale of the process. Even in the case of by enzymes. The rate of degradation depends on the specificity of involved enzymes. The rapidly-degraded polymers in the presence of highly-active enzymes, the degradation rate can be complication is connected with the time-scale of the process. Even in the affected of by enzymatic the decay of degradation enzymatic activity. case of rapidly-degraded polymers in the presence of highly-active enzymes, the degradation It is known that common extracellular proteinases of mammals, such as chymotrypsin A and rate can be elastase, affectedboth by the decay of enzymatic serine proteinases which activity. are active towards the bonds formed with hydrophobic neutral that amino acids, are at least two proteinases orders of magnitude less active the charged A and It and is known common extracellular of mammals, suchtowards as chymotrypsin poly(α-amino comparatively lyposomal endopeptidase cathepsin B (thiol proteinase with and elastase, both serineacids) proteinases whichtoare active towards the bonds formed with hydrophobic broad specificity) [38]. Papain, a thiol proteinase of plant origin, is known as the analog of cathepsin B neutral amino acids, are at least two orders of magnitude less active towards the charged regarding its activity towards the bonds formed between different α-amino acids. poly(α-amino acids) comparatively to lyposomal endopeptidase cathepsin B (thiol proteinase with In our work, the biodegradation of prepared particles was studied using a model enzyme system broad specificity) [38]. plasma Papain,under a thiol proteinaseconditions. of plant origin, is known analog of cathepsin and human blood physiological Papain was chosen as as athe model proteolytic B regarding its The activity towardsprocess the bonds formedasbetween different α-amino enzyme. degradation was studied a function of free amino acidsacids. accumulation in Inthe our work,mixture. the biodegradation ofFigure prepared was studied a model enzyme reaction As it is seen from 8, the particles degradation of both PGlu using and PPhe catalyzed with papain occurred simultaneously. As expected, in the case of copolymer with shorter blocks, system and human blood plasma under physiological conditions. Papain was chosen as a model the degradation was achieved faster. In this case, the of polymer proteolytic enzyme. curve The plateau degradation process was studied asdegradation a function of free particles amino acids was practically finished after 45 days. At the same time, the degradation of polypeptide with longer accumulation in the reaction mixture. As it is seen from Figure 8, the degradation of both PGlu and chain blocks reached the plateau after approx. 60 days of degradation. PPhe catalyzed with papain occurred simultaneously. As expected, in the case of copolymer with In vitro degradation of the particles in blood plasma was less effective, and even after two shortermonths blocks,thethe degradation was achieved faster. thisactivity case, the of total degradation curve was notplateau achieved. It can be related to the In lower and,degradation probably, polymer particles practically finished after At process. the same time,difference the degradation of partial plasmawas enzyme inactivation during so long45 an days. incubation Another of the degradation physiological medium was the the mostplateau effectiveafter degradation of 60 thedays PGlu of block compared polypeptide with in longer chain blocks reached approx. degradation. to the PPhe one, which can be related to the differences in the biocatalyst nature present in the systems. In vitro degradation of the particles in blood plasma was less effective, and even after two In any case, it can be concluded that the degradation of poly(amino acid)-based polymersomes at months the total degradation was not achieved. It can be related to the lower activity and, probably, pH 7.4 and 37 ˝ C is not quick, and takes several weeks. partial plasma enzyme inactivation during so long an incubation process. Another difference of the degradation in physiological medium was the most effective degradation of the PGlu block compared to the PPhe one, which can be related to the differences in the biocatalyst nature present in the systems. In any case, it can be concluded that the degradation of poly(amino acid)-based polymersomes at pH 7.4 and 37 °C is not quick, and takes several weeks.

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Figure Figure8.8. Accumulation Accumulation of of free free amino amino acids acids during during the the biodegradation biodegradation process process of ofPGlu-b-PPhe PGlu-b-PPhe ˝ polymersomes conditions(pH (pH7.4, 7.4,3737 degradation GP1 using papain; polymersomes at at physiological physiological conditions °C).C). (A)(A) degradation of of GP1 using papain; (B) (B) degradation GP3 using papain; and in vitro degradation GP3 using blood serum plasma. degradation GP3 using papain; and (C)(C) in vitro degradation of of GP3 using blood serum plasma.

3.4. 3.4.Surface SurfaceModification Modification The Thesurface surfacefunctional functionalgroups groupsof ofthe thediscussed discussednanoparticles nanoparticlesopen openthe thewide widepossibility possibilityfor fortheir their modification. modification.ItItisisknown knownthat thatproteins proteinsand andpeptides peptidesare areoften oftenused usedas asvector vectorligands ligandsfor forthe thetargeted targeted drug drugdelivery delivery[39]. [39].In Inthis thiswork workthe thelabile labileenzyme enzymeribonuclease ribonucleaseAAwas wasused usedfor forfunctionalization functionalizationof of GP2 GP2nanoparticles. nanoparticles.The Theimmobilization immobilizationcapacity capacitywas wasequal equaltoto0.7 0.7mg/mg mg/mgof ofparticles. particles.The Theevaluation evaluation of of the the enzyme enzyme activity activity was was performed performed using using thecytidine-2’,3’-cyclophosphate thecytidine-2’,3’-cyclophosphate as as aa specific specific low low molecular TheThe comparison of activity of freeofand bound biocatalyst allowed molecularweight weightsubstrate. substrate. comparison of activity free and forms boundofforms of biocatalyst the conclusion that the applied biofunctionalization did not contribute to the enzyme allowed the conclusion that themethod appliedofmethod of biofunctionalization did not contribute to the inactivation (Table 3).(Table Additionally, the results modification of GP3 with α-chymotrypsin can be enzyme inactivation 3). Additionally, theon results on modification of GP3 with α-chymotrypsin found the Supplementary data (Table S1).(Table The activity this enzyme studied as described can beinfound in the Supplementary data S1). Theofactivity of thiswas enzyme was studied as elsewhere described [40]. elsewhere [40]. Table Table3.3.Kinetic Kineticparameters parametersof ofcytidine-2’,3’-cyclophosphate cytidine-2’,3’-cyclophosphatehydrolysis hydrolysiscatalyzed catalyzedby byribonuclease. ribonuclease.

Biocatalyst form Biocatalyst form

Activity, µmol·min ´1 Activity, µmol¨ min´1 ¨ mg·mg

Free ribonuclease A Free ribonuclease A Immobilized ribonuclease Immobilized ribonuclease A A

-1

2.4

2.4 2.22.2

-1

mM KKMM, ,mM 27

27 18 18

3.5. Encapsulation of Model Compounds and Cell Experiments 3.5. Encapsulation of Model Compounds and Cell Experiments Loading of model compounds, namely, bromophenol blue and rhodamine 6g, into Loading of model compounds, namely, bromophenol blue and rhodamine 6g, into polymersomes polymersomes was carried out to prove the applicability of these kinds of particles as potential was carried out to prove the applicability of these kinds of particles as potential nanocontainers for nanocontainers for drug delivery. drug delivery. The encapsulation efficiency of bromophenol blue was in the range 8%–21% and depended on The encapsulation efficiency of bromophenol blue was in the range 8%–21% and depended on the process conditions. The encapsulation efficiency remained the same (about 20%) if the initial dye the process conditions. The encapsulation efficiency remained the same (about 20%) if the initial concentration did not exceed 1.0 mg/mL. In contrast, this value (%) linearly decreased to 8% if the initial concentration was increased up to 2.0–2.5 mg/mL (Figure S2 of Supplementary Material). The

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dye concentration did not exceed 1.0 mg/mL. In contrast, this value (%) linearly decreased to 8% if thePolymers initial concentration was up to 2.0–2.5 mg/mL S2 of Supplementary Material). 8, 212 11 model of 14 amount of2016, encapsulated dye increased (μg) reached the plateau, when(Figure the initial concentration of the The amount of above encapsulated dye The (µg)maximum reached the plateau, when dye the initial of the compound was 1.0 mg/mL. amount of loaded was 0.8concentration mg/mg of particles amount of encapsulated dye (μg) reached the plateau, when the initial concentration of the model model compound was above 1.0 mg/mL. The maximum amount of loaded dye was 0.8 mg/mg of (Figure S3 of Supplementary Material). Qualitatively, the approval of dye encapsulation into compound was above 1.0 mg/mL. The maximum amount of loaded dye was 0.8 mg/mg of particles particles (Figure S3 of Supplementary Material). Qualitatively, the approval of dye encapsulation into polymersomes was shown by TEM images (Figure 9). The native polymersomes have a light core, (Figure S3 ofwas Supplementary Material).(Figure Qualitatively, the approval of dye encapsulation into polymersomes shown by TEM 9).toThe native polymersomes light core, while while the loaded nanovesicles are images dark inside due bromophenol increasinghave theira inner electronic polymersomes was shown by TEM images (Figure 9). The native polymersomes have a light core, the loaded nanovesicles are dark inside due to bromophenol increasing their inner electronic density. density. while the loaded nanovesicles are dark inside due to bromophenol increasing their inner electronic density.

Figure of of GP2 polymersomes loaded withwith model dye dye (bromophenol blue):blue): (A) Figure 9. 9. TEM TEMimages images GP2 polymersomes loaded model (bromophenol 9. TEM images of 16%; GP2 polymersomes loaded with model dye (bromophenol blue): (A) encapsulation efficiency 16%; andand (B) encapsulation efficiency 21%. (A)Figure encapsulation efficiency (B) encapsulation efficiency 21%. encapsulation efficiency 16%; and (B) encapsulation efficiency 21%.

To test the biocompatibility and cytotoxicity of PGlu-b-PPhe polymersomes, MTT assay using ToTo testtest thethe biocompatibility and cytotoxicity of PGlu-b-PPhe polymersomes, MTT assay using HEK biocompatibility cytotoxicity of PGlu-b-PPhe MTT assay using HEK and Сaco-2 cell lines wasand performed. The suspensionspolymersomes, on PGlu62-b-PPhe 82 (GP2) and and Caco-2 cell lines was performed. The suspensions on PGlu -b-PPhe (GP2) and PGlu -b-PPhe 62 82 117 81 HEK and Сaco-2 cell lines was performed. The suspensions on PGlu 62-b-PPhe82 (GP2) and PGlu117-b-PPhe81 (GP3) at four different concentrations, ranging from 0.05 to 0.50 mg/mL, were (GP3) at117four different concentrations, ranging from 0.05 to 0.50 mg/mL, were incubated with cells PGlu -b-PPhe 81 (GP3) at four different concentrations, ranging from 0.05 to 0.50 mg/mL, were incubated with cells within 48 h. According to the results illustrated in Figure 10 for particles within 48 h. with According to the48results illustrated in Figure for particles prepared longer incubated cells within h. According to the results 10 illustrated in Figure 10 forfrom particles prepared from longer chain polymer no cytotoxicity was observed during the experimental time at chain polymer no cytotoxicity was observed during the experimental time at all tested concentrations. prepared from longer chain polymer no cytotoxicity was observed during the experimental time at all tested concentrations. The results obtained for the particles prepared from polymer with shorter The obtained for the prepared polymerprepared with shorter PGlu chain allresults tested concentrations. Theparticles results obtained forfrom the particles from polymer withabsolutely shorter PGlu chain absolutely coincided with those obtained for PGlu117-b-PPhe81-based polymersomes. coincided with those obtained for with PGluthose polymersomes. PGlu chain absolutely coincided obtained for PGlu 117-b-PPhe81-based polymersomes. 117 -b-PPhe 81 -based

(A) (A)

(B) (B)

Figure10.10.Viability Viability ofHEK HEK (A) and and Caco-2 (B) (B) cells cultured in medium containing different Figure Figure 10. Viability of of HEK (A) (A) and Caco-2 Caco-2 (B) cells cells cultured cultured in in medium medium containing containing different different concentrations of GP3 polymersomes. concentrations of GP3 polymersomes.

To investigate the penetration of particles obtained into the cells, the fluorescent dye rhodamine To investigate the penetration of particles obtained into the cells, the fluorescent dye rhodamine investigate the penetration of particles obtained and intoincubated the cells, the fluorescent rhodamine 6g To was encapsulated into PGlu-b-PPhe polymersomes with the Caco-2dye cells within 6g was encapsulated into PGlu-b-PPhe polymersomes and incubated with the Caco-2 cells within 6gfour washours. encapsulated into PGlu-b-PPhe polymersomes and incubated withThe thecoloration Caco-2 cells within The visualization of cells was done by fluorescent microscopy. of inner four hours. The visualization of cells was done by fluorescent microscopy. The coloration of inner four hours. Thecells visualization of cells was done by fluorescent microscopy. coloration of inner space space of the with encapsulated dye proved the cellular uptake of The developed polymersomes space of the cells with encapsulated dye proved the cellular uptake of developed polymersomes of (Figure the cells11). with encapsulated dye proved the cellular uptake of developed polymersomes (Figure 11).

(Figure 11).

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Figure Figure 11. 11. Bright-field Bright-field (A) (A) and and fluorescent fluorescent (B) (B) images images of of Caco-2 Caco-2 cell cell containing containing GP3 GP3 particles particles stained stained with rhodamine 6g. with rhodamine 6g.

4. Conclusions 4. Conclusions The The preparation preparation of of polymersomes polymersomes from from amphiphilic amphiphilic block-copolymer block-copolymer of of glutamic glutamic acid acid and and phenylalanine studied. The Thestructures structures obtained possessed self-assembled membrane, phenylalanine was studied. obtained possessed thethe self-assembled membrane, the the surface capable for biofunctionalization, and the core appropriate for encapsulation of hydrophilic surface capable for biofunctionalization, and the core appropriate for encapsulation hydrophilic compounds. compounds. The diameter of prepared polymersomes depended on pH of the the solution solution used used for for self-assembly, self-assembly, as as well well as as on on the the length length of of the thehydrophobic hydrophobicblock. block. Particularly, Particularly, the the mean mean size size of of polymersomes was managed to vary from 60 to 350 nm depending on the conditions of preparation polymersomes was managed to vary from 60 to 350 nm depending on the conditions of preparation and chain length. SuchSuch important features of developed polymersomes as the absence and on onthe thepolymer polymer chain length. important features of developed polymersomes as the of cytotoxicity and biodegradability, easy possibilities to functionalize the polymersome surface with absence of cytotoxicity and biodegradability, easy possibilities to functionalize the polymersome protein its inactivation, to encapsulate the hydrophilic compound inside the surface without with protein without the its capability inactivation, the capability to encapsulate the hydrophilic nanoparticles and to them into thetocells, make thisinto kindthe of cells, polymer nanoconstruction quite compound inside thedeliver nanoparticles and deliver them make this kind of polymer attractive for further development of specifically-targeted drug delivery formulations. nanoconstruction quite attractive for further development of specifically-targeted drug delivery formulations.

Supplementary Materials: Supplementary Materials can be found at www.mdpi.com/2073-4360/8/6/212/s1. Supplementary Materials: Supplementary Materials can be found at www.mdpi.com/2073-4360/8/6/212/s1. Acknowledgments: This work was supported by grant of Russian Scientific Foundation (#14-50-00069). The participation in this work of Anastasiia Hubinaby(monthly supported by postdoc program from Acknowledgments: This work was supported grant of salary) Russianwas Scientific Foundation (#14-50-00069). The St. Petersburg State University (#12.50.1193.2014). Chromatographic experiments and NMR experiments were participation in this work of Anastasiia Hubina (monthly salary) was supported by postdoc program from St. carried out in Chemical Analysis and Materials Research Centre and Resource Center for Magnetic Resonance Petersburg State State University (#12.50.1193.2014). Chromatographic and NMR experiments were (Saint-Petersburg University). Cell experiments were performed experiments using the equipment of the Research Center carried out in Chemical Analysis Research Centre and Resource Center for Magnetic Resonance on Development of Molecular andand CellMaterials Technologies (Saint-Petersburg State University). Also the authors thank Thomas Scheper and Antonina Lavrentieva (Institute of Technical Chemistry, Leibniz University, (Saint-Petersburg State University). Cell experiments were performed using the equipment of theHannover, Research Germany) their help with visualization experiments. Center onfor Development of cell Molecular and Cell Technologies (Saint-Petersburg State University). Also the authorsContributions: thank Thomas Scheper Antonina Lavrentieva (Institute of Technical Chemistry, University, Author Evgeniaand Vlakh developed the concept, planned the experiments andLeibniz analyzed the data; Anastasiia Pogodaev performedexperiments. the experiments on polymer synthesis, preparation Hannover,Ananyan Germany)and for Aleksander their help with cell visualization and modification of particles; Anastasiia Hubina characterized the particles with DLS and TEM methods; AuthorVolokitina Contributions: Evgenia Vlakh developed the concept,Natalia planned the experiments andthe analyzed the data; Mariia preformed the biodegradation experiments; Zashikhina performed experiments on dye encapsulation; Natalia Zashikhina Pogodaev and Vladimir Sharoykothe performed the cell Tatianapreparation Tennikova Anastasiia Ananyan and Aleksander performed experiments on experiments; polymer synthesis, managed the project; Evgenia Vlakh and Tatianathe Tennikova wrote paper. authors have and modification ofAnastasiia particles; Hubina, Anastasiia Hubina characterized particles withthe DLS andAll TEM methods; given approval for the final version of the manuscript. Mariia Volokitina preformed the biodegradation experiments; Natalia Zashikhina performed the experiments Conflicts of Interest: The authorsZashikhina declare no and conflict of interest. on dye encapsulation; Natalia Vladimir Sharoyko performed the cell experiments; Tatiana Tennikova managed the project; Anastasiia Hubina, Evgenia Vlakh and Tatiana Tennikova wrote the paper. All authors have given approval for the final version of the manuscript. Abbreviations The following abbreviations are used in thisnomanuscript: Conflicts of Interest: The authors declare conflict of interest. GPC Gel permeation chromatography Abbreviations TEM Transmission electron microscopy NCA N-carboxyanhydrides The following abbreviations are used in this manuscript: ROP Ring-opening polymerization GPC: Gel permeation chromatography HEXA Hexylamine TEM: Transmission electron microscopy DMF Dimethylformamide DMSO Dimethyl sulfoxide NCA: N-carboxyanhydrides TFMSA Trifluoromethane sulfonic acid ROP: Ring-opening polymerization TFA Trifluoroacetic acid HEXA: Hexylamine NHS N-hydroxysuccinimide DMF: Dimethylformamide DMSO: Dimethyl sulfoxide

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1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide Nuclear magnetic resonance Dynamic light scattering High performance liquid chromatography Human embryonic kidney cells Human epithelial colorectal adenocarcinoma cells

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