Smart Polymeric Nanocarriers of Met-enkephalin - Biomacromolecules

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Smart Polymeric Nanocarriers of Met-enkephalin Roza Szweda, Barbara Trzebicka, Andrzej Dworak, Lukasz Otulakowski, Dominik Kosowski, Justyna Hertlein, Emi Radoslavova Haladjova, Stanislav Miletiev Rangelov, and Dawid Szweda Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b00725 • Publication Date (Web): 13 Jul 2016 Downloaded from http://pubs.acs.org on July 19, 2016

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Smart Polymeric Nanocarriers of Met-enkephalin Roza Szweda,† Barbara Trzebicka,†* Andrzej Dworak,† Lukasz Otulakowski,† Dominik Kosowski,† Justyna Hertlein,† Emi Haladjova,‡ Stanislav Rangelov,‡ and Dawid Szweda† †

Centre of Polymer and Carbon Materials, Polish Academy of Sciences, M. Curie-Sklodowskiej

34, Zabrze, 41-819, Poland ‡

Institute of Polymers, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., bl. 103-A, Sofia,

1113, Bulgaria

ABSTRACT

This study describes a novel approach to polymeric nanocarriers of the therapeutic peptide metenkephalin based on the aggregation of thermoresponsive polymers. Thermoresponsive bioconjugate poly((di(ethylene glycol) monomethyl ether methacrylate)-ran-(oligo(ethylene glycol) monomethyl ether methacrylate) is synthesized by AGET ATRP using modified metenkephalin as a macroinitiator. The abrupt heating of bioconjugate water solution leads to the self-assembly of bioconjugate chains and the formation of mesoglobules of controlled sizes. Mesoglobules formed by bioconjugates are stabilized by coating with crosslinked two-layer shell via nucleated radical polymerization of N-isopropylacrylamide using a degradable cross-linker. The targeting peptide RGD, containing the fluorescence marker carboxyfluorescein, is linked to a nanocarrier during the formation of the outer shell layer. In the presence of glutathione, the whole shell is completely degradable and the met-enkephalin conjugate is released. It is

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anticipated that precisely engineered nanoparticles protecting their cargo will emerge as the nextgeneration platform for cancer therapy and many other biomedical applications.

KEYWORDS: bioconjugates, met-enkephalin, nanocarriers, aggregation, thermoresponsiveness

1. INTRODUCTION Nanotechnology has become a rapidly growing medical interest as it promises to solve a number of issues associated with conventional therapeutic agents, including their often poor water solubility, lack of targeting capability, nonspecific distribution, systemic toxicity, and low therapeutic index. Over the past several decades, remarkable progress has been made in the development and application of engineered nanoparticles1-7 with optimal sizes, shapes, and surface properties to increase solubility, prolong circulation half-life, improve biodistribution, and reduce the immunogenicity of their payloads. Recently, peptide-based therapy has been extensively studied and utilized for the treatment of various diseases, including cancers.8-11 Their wider application is limited by their poor oral bioavailability due to rapid degradation in the gut or in the blood and excretion from the organism, which results in a low therapeutic effect.12,13 Moreover, their frequently high molar mass and chemical composition, which is often a cocktail of charges and hydrophobic groups, hamper cellular uptake. The problems of delivery and the controlled release of anticancer peptides, constituting one of the main problems of contemporary pharmaceutic treatment,12,13 can be overcome by the application of nanocarriers where drugs are entrapped physically or chemically. Nanocarriers can be favorably delivered into tumors by targeting ligands (e.g., small organic molecules, peptides,


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antibodies, and nucleic acids) which specifically target cancerous cells through selective binding to the receptors overexpressed on their surface.14-17 A prominent example for targeting is short peptide RGD (arginine-glycine-aspartic acid),16 which is present in many extracellular matrix components such as fibronectin and vitronectin. RGD is specifically recognized by the overexpressed integrin receptors on the tumor cells, thus ensuring the delivery of drugs mainly to the tumor by specific ligand-receptor interactions.16 One of the most important, clinically relevant peptides is met-enkephalin (tyrosine-glycineglycine-phenylalanine-methionine), an endogenous opioid peptide, which has anti-tumor activity against a diverse range of cancers.18,19 Unlike chemotherapy, met-enkephalin does not directly destroy cancer cells and is not cytotoxic. It does, however, halt the growth of the cells and is thought to allow immunological mechanisms (e.g., macrophages, natural killer cells) to accomplish the task of destroying cancerous cells. The anti-cancer effects of met-enkephalin after parenteral administration in humans has significant limitations because of low drug concentrations due to the poor aqueous solubility of the peptide. It was demonstrated that metenkephalin encapsulated in nanocarriers created by the amphiphilic polymer quaternary ammonium palmitoyl glycol chitosan, forming ‘molecular envelopes’, is orally bioavailable and active in murine models of pancreatic cancer.20 Thermoresponsive polymers belong to the group of “smart polymers” which exhibit phase transition in water. Above a certain temperature, thermoresponsive polymers precipitate,21-23 while in dilute solution, their chains aggregate to particles called mesoglobules of sizes ranging from tens to thousands of nanometers.24,25 Mesoglobules’ sizes and structure can be tuned by controlling many parameters of the aggregation process such as the concentration of polymer


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solution, heating rate, polymer molar mass, polymer composition, the presence of surfactants and salts.25-27 The conjugates of peptides with thermoresponsive polymers also display self-assembly into mesoglobules. Only a few works concerning this phenomenon with the description of the behavior of model peptides conjugated with poly(N-isopropylacrylamide) (PNIPAM)28-30 and poly(di(ethylene glycol) methyl ether methacrylate)31 have been published. The mesoglobules of thermoresponsive polymers disaggregate below the transition temperature, which significantly limits their practical usage. However, they can be stabilized, when covered by a cross-linked shell obtained by nucleated radical polymerization27,32,33 or intermolecular crosslinking, as shown recently by Dworak et al.34 Mesoglobules were mainly stabilized by covering them with crosslinked shells using nucleated radical polymerization of N-isopropylacrylamide (NIPAM)27 or 2-hydroxyethylmethacrylate.32 Mesoglobules of poly(glycidol-co-ethyl glycidyl carbamate)s,27 poly(methoxydiethyleneglycol methacrylate),33 poly(2-isopropyl oxazoline)35 and PNIPAM32 were used as core templates. It was shown that the removal of polymer chains from the particle interior can be reached by diffusion32 or by shell degradation.36 Here, we described the procedure leading to the effective synthesis of targeted nanocarriers of met-enkephalin. An approach proposed in this work is based upon self-assembly of the bioconjugate consisting of met-enkephalin linked with the chains of a thermoresponsive polymer to mesoglobules. The mesoglobules were stabilized by covering them with cross-linked shells using nucleated radical polymerization of N-isopropylacrylamide in the presence of N,N’bis(acryloyl)cystamine. During the coating process, the RGD targeting ligand was introduced to the outer shell of nanocarrier. The presence of a disulfide bond in the cross-linker structure


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enabled degradation of the nanocarrier shell in the presence of glutathione, which resulted in the release of met-enkephalin conjugate. The presented synthetic strategy opens up a route to the new type of polymeric peptide nanocarriers for targeted anti-cancer therapy. 2. MATERIALS AND METHODS 2.1. Materials. Tentagel S RAM resin was purchased from Rapp Polymere (Germany). Dansyl NovaTag™ resin and Fmoc-Lys(Mtt) (>98%) were purchased from Merck. Amino acids (FmocGly-OH, (98.0%), Fmoc-Phe-OH, (98%), Fmoc-Met (98%), Fmoc-Tyr(tBu) (98%), FmocArg(Pbf)-OH (98%), Fmoc-Asp(OtBu) (98%)), 2-bromopropionic acid (99%), (5,6)carboxyfluorescein (CF, ≥95%), acrylic acid (99%), phenol (PhOH, 99%), 1,2-ethanedithiol (EDT, >98%), triisopropylsilane (TIS, 99%), CuBr2 (99%), tris(2-pyridylmethyl)amine (TPMA, 98%), ʟ-ascorbic acid (99%), methyl 2-bromopropionate (98%), di(ethylene glycol) monomethyl ether methacrylate (D, DEGMA-ME, 95%), oligo(ethylene glycol) monomethyl ether methacrylate (O300, OEGMA-ME300, Mn = 300 g/mol), ethyl (hydroxyimino)cyanoacetate triisopropylsilane (Oxyma, 97%), N,N-diisopropylcarbodiimide (DIC, 99%), trifluoroethanol (TFE, 99%), 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP,>99%), trifluoroacetic acid (TFA, 99%), piperidine (99%), water (CHROMASOLV® for HPLC), acetonitrile (CHROMASOLV® for HPLC),

N-isopropylacrylamide

(NIPAM,

≥99%),

N,N′-bis(acryloyl)cystamine

(BAC,

BioReagent), potassium persulfate (KPS, ≥99.0%), sodium dodecyl sulfate (SDS, 98%), trypsin from bovine pancreas suitable for protein sequencing, and ʟ-glutathione reduced (≥98.0%) were purchased from Sigma–Aldrich. Methanol (MeOH, 99.8%), dichloromethane (DCM, 99.8%), N,N-dimethylacrylamide (DMF, 99.8%), diethyl ether (pure p.a.), n-buthanol (nBuOH, pure p.a.), phenol (pure p.a.) were obtained from Avantor Performance Materials (Poland).


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Spectra/Por® dialysis membranes cut-off 6000-8000 Da and 50000 Da were obtained from Spectrum Laboratories (USA). 2.2. Peptides. Met-enkephalin (Br-MEDns, 2-bromopropionate-tyrosine-glycine-glycinephenylalanine-methionine-dansyl) and

CFRGD-KAAm

(carboxyfluorescein-arginine-glycine-

aspartic acid-lysine(acrylamine)) were synthesized according to the Fmoc strategy of solid phase peptide synthesis using DIC and Oxyma as activators.37 The details of peptides synthesis are described in the Supporting Information. 2.3. Synthesis of P(D-ran-O300)-MEDns Bioconjugate. Conjugate of met-enkephalin and poly((di(ethylene

glycol)

monomethyl

ether

methacrylate)-ran-(oligo(ethylene

glycol)

monomethyl ether methacrylate)) (P(D-ran-O300)-MEDns) was obtained by AGET ATRP with peptide as an initiator. Synthesis was performed using a method similar to that reported previously by Averick et al.38 In a Schlenk flask purged with argon (di(ethylene glycol) monomethyl ether methacrylate (0.600 mL, 3.252 mmol), oligo(ethylene glycol) monomethyl ether methacrylate (0.400 mL, 0.1394 mmol), Met-enkephalin modified by 2-bromopropionic acid as an initiator (23.69 mg, 23 µmol), CuBr2 (51.9 mg, 230 µmol), TPMA (74.2 mg, 255 µmol), methanol (1 mL) and water (0.5 mL) were added and degassed by 3 freeze-thaw-pump cycles. Parallel ascorbic acid (4.0 mg, 23 µmol) was dissolved in water (0.1 mL) and purged with argon for 30 minutes. An ascorbic acid solution was then added to the Schlenk flask with other reagents (after last freeze-thaw-pump cycle) and the reaction was conducted for 4.5 h at RT. The conjugate was purified by dialysis through a membrane (cut-off 6000-8000 Da) against water/acetone solution (1:1) for 2 days, followed by pure acetone for 3 days. 2.4. Synthesis of P(D-ran-O300) Model Copolymer. Model copolymer poly((di(ethylene glycol) monomethyl ether methacrylate)-ran-(oligo(ethylene glycol) monomethyl ether


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methacrylate)) (P(D-ran-O300)) was obtained by AGET ATRP using methyl 2-bromopropionate as initiator. Polymerization procedures were the same as for bioconjugate and reagent’s molar ratio

was

[DEGMA-ME]/[OEGMA300]/[initiator]/[CuBr2]/[TPMA]/[ascorbic

acid]

=

600/260/1/10/11/1. Polymerization was stopped after 1.5 h. The copolymer was purified by dialysis through a membrane (cut-off 6000-8000 Da) against water/acetone solution (1:1) for 2 days, followed by pure acetone for 3 days. 2.5. Formation of Mesoglobules. For the preparation of mesoglobules from bioconjugate or model copolymer their water solutions at different initial concentrations (c = 0.1, 0.2, 0.5 or 1.0 g/L) were prepared. These aqueous solutions were subjected to gradual or abrupt heating in the presence or absence of an anionic surfactant (SDS). The surfactant to polymer weight ratio (s/p) was fixed at 0.2.32 All of the solutions used were chilled to 4°C before use. In a gradual heating protocol, 4 mL of certain solutions were subjected to a gradual increase of temperature from 18°C to 55°C using a thermocontroller. In the abrupt heating protocol, 2 mL of P(D-ran-O300) or P(D-ran-O300)-MEDns solutions were placed in a preheated at 55°C bath. 2.6. Coating of Bioconjugate Mesoglobules. Mesoglobules obtained by abrupt heating at fixed concentration of 0.2 g/L, in presence of SDS (s/p = 0.2) were used for the coating procedure. After annealing the solutions at 55°C for 30 min, the temperature was raised to 65°C. Under nitrogen and vigorous stirring, 0.18 mL of mixed aqueous solution of NIPAM (9.55 mmol/L) and BAC (0.4 mmol/L) were added to 4 mL aqueous dispersions of mesoglobules at 65°C. After 30 min, the initiator KPS (0.1 mL, 0.1 M solution) was introduced into the mixture and the reaction was allowed to proceed for 3 h to form the first layer of the shell. Next, 0.1 mL of mixed water solution containing NIPAM (5.3 mmol/L), BAC (0.25 mmol/L), CFRGD-KAAm (0.01 mmol/L) was introduced to the dispersion and 0.05 mL KPS (0.1 M) was added. After 2 h,


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the reaction was terminated and the coated particles were subjected to dialysis against water for 16 h at room temperature. Spectra/Pore® dialysis membrane with MWCO 50000 was used to remove the unreacted monomers, initiator and cross-linker. The size of the coated particles was measured at 65 and 25°C. 2.7. Degradation of Nanoparticles. For degradation experiments, 0.10 mL of pH = 7.4 buffer was added to 1 mL of dispersion containing nanoparticles. Next, 30 mg of reduced glutathione was added. Probes were incubated in a water bath at 37°C. At preselected time periods, DLS measurements of the dispersions were performed at 37°C below the TCP of bioconjugate. 2.8. Methods. 2.8.1. Proton Nuclear Magnetic Resonance (1H NMR). 1H NMR spectra of the bioconjugate and the model copolymer were recorded on a Bruker Ultrashield spectrometer operating at 600 MHz in CDCl3 with TMS as reference. 2.8.2. Gel Permeation Chromatography. The molar masses and molar mass dispersities of the bioconjugate and model copolymer were determined using gel permeation chromatography (GPC-MALLS). The Dn-2010 RI differential refractive index detector (WGE Dr. Bures) and a HELEOS multi-angle laser light scattering detector (Wyatt Technologies) were used. GPC was performed in DMF at 45°C with 5 mmol/L LiBr at a nominal flow rate of 1 mL/min using a set of columns: Gram 100 Å, Gram 1000 Å, Gram 3000 Å (Polymer Standard Service). The results were evaluated using ASTRA 5 software (Wyatt Technologies). The refractive index increment (dn/dc) of P(D-ran-O300)-MEDns (dn/dc = 0.0523 mL/g) and P(D-ran-O300) (dn/dc = 0.0513 mL/g) were independently measured in DMF using a SEC-3010 differential refractive index detector (WGE Dr. Bures) and used to calculate the average molar masses of the copolymers.


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2.8.3. Cloud Point Measurement. The cloud points (TCP) of P(D-ran-O300)-MEDns and P(D-ranO300) were determined on a Jasco V-530 UV–vis spectrophotometer with a cuvette thermostatted by a Medson MTC-P1 Peltier thermocontroller. The transmittances of the 1 g/L solutions/dispersions were monitored at λ = 550 nm as a function of temperature. The cloud points refer to the inflection points of the transmittance curves. 2.8.4. Dynamic Light Scattering. DLS measurements were performed on a Brookhaven BI-200 goniometer with vertically polarized incident light of wavelength λ = 632.8 nm supplied by a HeNe laser operating at 35 mW and equipped with a Brookhaven BI-9000 AT digital autocorrelator. The scattered light was measured for aqueous copolymer solutions/dispersions at concentrations of 0.1, 0.2, 0.5, and 1 g/L at an angle of 90 °. The autocorrelation functions were analyzed using the constrained regularized CONTIN method to obtain distributions of relaxation rates (Γ). The latter provided distributions of the apparent diffusion coefficient (D = Γ/q2), where q is the magnitude of the scattering vector, q = (4πn/λ)sin(θ/2) and n is the refractive index of the medium. The apparent hydrodynamic radius (ܴ୦ଽ଴ ) was obtained from the Stokes-Einstein equation (Equation 1). ௞்

ܴ୦ଽ଴ = ଺గఎ஽

(1)

for θ = 90 ° where k is the Boltzmann constant, η is the viscosity of water at temperature T, and D is the apparent diffusion coefficient. The dispersity of particle sizes was given as ߤଶ /߁ത ଶ , where ߁ത is the average relaxation rate and µ2 is its second moment. The cloud point temperatures refer to the inflection points of dependence of the ܴ୦ଽ଴ versus temperature. 2.8.5. Static Light Scattering. SLS measurements were conducted using the same light scattering set-up as in DLS studies. Because the size of the mesoglobules depends on the concentration of the polymer in the solution below the phase transition, here to preserve the sizes


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of the formed aggregates, the initial 0.2 g/L dispersion of the bioconjugate at 65°C was diluted with water of the same temperature to prepare the samples of various mesoglobules’ concentrations. The measurements were carried out at angles from 40 to 140° every 10 degree. The SLS data were analyzed with the BIC Static Light Scattering software using the Zimm plot. Information on the weight-average molar mass of mesoglobule (Mw, mesoglobule) was obtained from the dependence of the quantity Kc/RΘ on the concentration, c and scattering angle, Θ. Here, K is the optical constant and RΘ is the Rayleigh ratio at Θ. 2.8.6. Fluorescence Spectroscopy. Fluorescence spectra were recorded on a Hitachi F-2500 fluorescence spectrophotometer at room temperature with excitation and emission appropriate for fluorophores - λex = 323 nm, λem = 520 nm for dansyl and λex = 496 nm, λem = 520 nm for carboxyfluorescein. The excitation and emission bandwidths were both 5 nm, the lamp voltage was 400 V. 2.8.7. Atomic Force Microscopy (AFM). Atomic Force Microscopy (AFM) was applied to visualize nanoparticles. The samples for AFM analysis were prepared by dropping 10 µL of nanoparticle solution in water on a silicon wafers cleaned by piranha solution and spin coated through 1 h with a rotation speed of 400 rpm/min. The measurements were performed using Multi-Mode with a NanoScope 3D controller (di-Veeco Instruments Inc., USA, CA), which was operated in tapping mode in air atmosphere with standard 125 nm single-crystal silicon cantilevers (Model TESP, Veeco Instruments Inc., USA). Images were obtained using a piezoelectric scanner. Data were evaluated by WSxM 5.0 software.39 3. RESULTS AND DISCUSSION The synthetic strategy presented in this work involved: (1) the synthesis of fluorescentlylabeled peptides met-enkephalin (clinically relevant drug) and RGD (targeting molecule) with


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additional functional groups able to initiate polymerization or undergo a copolymerization process, (2) the synthesis of thermoresponsive P(D-ran-O300)-met-enkephalin conjugate (P(Dran-O300)-MEDns), (3) studying temperature-induced aggregation of the bioconjugate to obtain appropriate sized particles, (4) covering mesoglobules with crosslinked shells using nucleated radical polymerization of N-isopropylacrylamide with simultaneous introducing RGD units and (5) study of nanoparticle degradation. The products/particles at each step of synthesis were characterized with appropriate techniques i.e. RP-HPLC, ESI-MS, GPC, DLS, AFM, fluorescence, UV-vis and 1H NMR. 3.1. Synthesis and Characterization of Peptides The met-enkephalin (Br-MEDns) sequence equipped with a fluorescent label and a bromine moiety (2-bromopropionate-tyrosine-glycine-glycine-phenylalanine-methionine-dansyl), which is able to initiate the atom transfer radical polymerization (ATRP) of (met)acrylic monomers,31 was synthesized on solid support Dansyl Nova Tag resin using Fmoc strategy, yielding peptides labeled with dansyl at its C-end.37 To the N-end of the last tyrosine residue, the 2bromopropionic acid was attached. The details of synthesis are described in the Supporting Information. The scheme of the reaction is shown in Scheme S1 (Supporting Information). The structure of the obtained met-enkephalin was confirmed by mass spectrometry analysis (Figure S1, Supporting Information). Its purity was 99%, as determined by RP-HPLC (Figure S2, Supporting Information). The chemical structure of the obtained met-enkephalin and its fluorescence spectra are shown in Figure 1A. To obtain fluorescently labeled

CFRGD-KAAm

targeting motif (carboxyfluorescein-arginine-

glycine-aspartic acid-lysine(acrylamide)) with acrylamide function, suitable to be connected to the outer part of nanocarrier, Fmoc strategy of solid phase peptide synthesis was applied37 as


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described in the Supporting Information (Scheme S2, Supporting Information). During synthesis, the ε-amine group of Fmoc-Lys(Mtt) was selectively deprotected in mild acidic conditions40 and reacted with acrylic acid containing double bonds that are active in the polymerization process. Carboxyfluorescein was coupled to the N-end of last arginine residue to make the fluorescent detection of peptides possible. The structure of the obtained peptides was confirmed by ESI-MS analysis (Figure S3, Supporting Information). Its purity was 99%, as determined by RP-HPLC (Figure S4, Supporting Information). The chemical structure of

CFRGD-KAAm

and its

Figure 1. Structures and fluorescence spectra of A) Br-met-enkephalinDns and B)

CFRGD-

fluorescence spectra are shown in Figure 1B.

KAAm. The fluorescence spectra of met-enkephalin and

CFRGD-KAAm

shown in Figure 1 evidenced

the characteristic maxima of emission and excitation spectra for applied fluorophores. They


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occur at λex = 323, λem = 520 for dansyl linked to met-enkephalin and λex = 496, λem = 520 for carboxyfluorescein attached to an RGD motif. ESI-MS, RP-HPLC and fluorescence analyses confirmed that the applied synthetic strategy led to well-defined, fluorescently-labeled peptide products containing bromide or acrylamide functions, respectively. The obtained met-enkephalin with a bromide group was used for initiation atom transfer radical copolymerization of OEGMAs monomers to obtain thermoresponsive bioconjugates.

The second peptide,

CFRGD-KAAm,

was used in

copolymerization with NIPAM in the process of formation of nanoparticles’ crosslinked shell. 3.2. Synthesis and Characterization of P(D-ran-O300)-met-enkephalin Conjugate P(D-ran-O300)-MEDns conjugate was synthesized by AGET ATRP (Scheme 1) using the modified method reported previously by Averick et al.38 The synthesis of bioconjugate was performed in methanol/water solution using met-enkephalin macroinitiator in the presence of the CuBr2/TPMA catalytic system and ascorbic acid as a reducing agent.

Scheme 1. The scheme of AGET ATRP copolymerization of DEGMA-MEDns and OEGMA300 with the use of a met-enkephalin macroinitiator.


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The bioconjugate number average molar mass was 93100 g/mol, as indicated by GPC. The GPC chromatogram of the P(D-ran-O300)-MEDns showed a monomodal molar mass distribution and low molar mass dispersity (Mw/Mn) of 1.23 (Figure 2A).

Figure 2. A) GPC-MALLS chromatogram (RI and LS traces) of P(D-ran-O300)-ME B) 1H NMR spectra of P(D-ran-O300)-MEDns (600 MHz, CDCl3). C) The fluorescence emission spectra of P(D-ran-O300)-MEDns and P(D-ran-O300) (10 mg/mL). The bioconjugate`s Mn value (93100 g/mol) was higher than the value calculated based on monomer/initiator molar ratio (Mn,th = 44000 g/mol). The difference could be caused by the high


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activity of the TPMA catalyst, which led to a high concentration of radicals and significant termination of the active centers at the first step of polymerization.38 Furthermore, according to the literature, the amide-based (peptide-based) initiators are a class of compounds which are problematic under ATRP reaction conditions.31,41 Polymers formed using these initiators have low dispersity, but considerably higher molar mass than theoretically assumed.42 In order to study the influence of the peptide segment on bioconjugate properties and its thermally induced aggregation the model (peptide free) copolymer P(D-ran-O300) with a molar mass similar to that of bioconjugate was synthesized by AGET ATRP. The GPC-MALLS chromatogram of model P(D-ran-O300) was monomodal and the molar mass dispersity (Mw/Mn) was 1.39 (Figure S5, Supporting Information). The number average molar mass of the P(D-ranO300) was 114000 g/mol. The structures and compositions of the bioconjugate (Figure 2B) and model copolymer (Figure S6, Supporting Information) were determined by 1H NMR in CDCl3. Signals: b,b’ (δ = 0.75÷1.1 ppm), a,a’ (δ = 1.7÷2.0 ppm), e,e’ (δ = 3.4 ppm), d,d’ (δ = 3.5÷3.8 ppm) and c,c’ (δ = 4.1 ppm) indicated protons originating from the P(D-ran-O300) copolymer chain. Peptide segment in the structure of bioconjugate was not visible in the 1H NMR spectrum (Figure 2B) because the quantity of protons coming from the P(D-ran-O300) chain was considerably greater than in the peptide segment. The compositions of the bioconjugate and model copolymer determined from the ratio of signals in their 1H NMR spectra revealed 67 mol % D and 33 mol % O300 for the bioconjugate, and 68 mol % D and 32 mol % O300 for P(D-ran-O300), which correspond well with the comonomer feed ratio (70 mol % D and 30 mol % O300, see experimental section).


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The presence of a met-enkephalin segment in the bioconjugate macromolecules was confirmed by fluorescence measurements. The fluorescence emission spectra of the bioconjugate and copolymer are compared in Figure 2C. The maximum values of emission (520 nm) and excitation (323 nm) for bioconjugate and met-enkephalin (Figure 1A and 2C) were observed at the same wavelength. The fluorescence of the bioconjugate originates specifically from the dansyl present at the end of peptide sequence confirming its attachment to copolymer chains. No fluorescence signal was observed for the model copolymer. 3.3. Thermoresponsiveness and Aggregation of Bioconjugate The thermoresponsive properties of polymers from the POEGMA family strongly depend on their composition;26 thus, the composition of bioconjugate was carefully chosen to maintain its phase transition temperature slightly above 37°C. TCP above physiological temperature ensures the disaggregation of bioconjugate mesoglobules in an organism at 37°C and thus fulfills the requirements of a drug formulation. The thermal behavior of aqueous solutions of P(D-ran-O300)-MEDns conjugate was monitored by UV-vis spectroscopy and light scattering to measure phase transition temperature and determine the possibility of controlling the sizes of mesoglobules formed at different conditions. The transmittance curves (Figure 3A) evidence temperature-induced phase transitions of bioconjugate. The presence of MEDns shifted TCP of bioconjugate by about 2°C to the lower temperatures (TCP = 38.5°C) in comparison with P(D-ran-O300) (TCP = 40.5°C), indicating that the peptide moiety increased the hydrophobicity of P(D-ran-O300) chains. It is known based on the behavior of other thermoresponsive polymers that the introduction of hydrophobic moieties to the thermoresponsive polymer chains causes the alteration of TCP and shifts the corresponding cloud point towards lower temperatures.43-45 The transitions for


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bioconjugate and model copolymer were rather narrow, spanning just 2-3°C. Small hysteresis was observed during cooling cycles of the samples (Figure 3A). As mentioned in the introduction, above TCP in dilute water solution thermoresponsive polymers aggregate into spherical particles called mesoglobules.46,47 Aggregation of the bioconjugate to mesoglobules is the first step necessary for the formulation of designed peptide nanocarriers. It is known that size of mesoglobules depends on several factors such as solution concentration, the molar mass and composition of the (co)polymer, the heating rate of the solution,26,46-50 and addition of the additives, with surfactants being the main focus of studies.27,51,52

Figure 3. A) Transmittance versus temperature of P(D-ran-O300)-MEDns and P(D-ran-O300) in water solutions (close symbols – heating, open symbols – cooling; concentration 1 g/L). B) Size


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distribution of P(D-ran-O300)-MEDns particles in water and water/SDS solutions subjected to abrupt and gradual heating (polymer concentration 0.2 g/L). To obtain the desired sizes of the bioconjugate’s mesoglobules, the study of aggregation depending on heating procedure, concentration, and addition of anionic surfactant sodium dodecyl sulfate (SDS) to water were performed. Two heating procedures were used for mesoglobules formation - gradual and abrupt heating of bioconjugate solutions at 0.1, 0.2, 0.5 and 1.0 g/L concentration in water and water containing SDS, maintaining the surfactant to polymer ratio equal 0.2. The obtained values of hydrodynamic radii are compared in Table 1. In pure water solution for both applied heating methods, the radii of mesoglobules depended strongly on bioconjugate concentration. It was observed that at the same concentration abrupt heating leads to bioconjugate`s mesoglobules of smaller sizes and seemingly narrower size distributions comparing to gradual heating (Figure 3B). Table 1. Hydrodynamic radii of mesoglobules formed by P(D-ran-O300)-MEDns.

Concentration[g/L] in water ૢ૙ in ࡾ‫[ ܐ‬nm] water/SDS

0.1 103 17

Abrupt heating 0.2 0.5 120 264 16

18

1.0 356

0.1 165

11

22

Gradual heating 0.2 0.5 250 457 21

21

1.0 516 17

Irrespective of heating procedure and concentration of the polymer solution, it was not possible to reduce mesoglobule radii in pure water below 100 nm. Therefore, based on the results reported in the literature51,52 and our experience,27,53 we performed the aggregation of bioconjugate in the presence of sodium dodecyl sulfate, which is known to decrease the sizes of aggregates of many thermoresponsive polymers. The applied ratio of SDS to bioconjugate s/p =


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0.2 was established taking into account studies performed previously for the aggregation of PNIPAM.32 The presence of SDS in solution resulted in the aggregation of bioconjugate chains to particles of several times smaller in diameter than those formed in pure water (Table 1). The size distribution dispersities for aggregates in water and in water/SDS remained nearly the same between 0.05 and 0.09 (Figure 3B). Similar behavior was observed for model copolymer (Figure S7, Supporting Information). SLS measurements were performed for mesoglobules obtained by abrupt heating of bioconjugate solution at fixed concentration of 0.2 g/L, in presence of SDS (s/p = 0.2). The SLS data were analyzed using the Zimm plot. The weight average molar mass of the mesoglobules, Mw,

mesoglobule,

at 65°C was 7.67×106 g/mol, which corresponded to approximately 67

macromolecules per mesoglobule, as calculated from the equation:

N agg =

M w,mesoglobule

(2)

M w,unimer

where Mw, unimer = 114500 g/mol was taken from GPC-MALLS. The obtained result Nagg = 67 corresponds to the average number of met-enkephaline molecules per mesoglobule, as there was one met-enkephalin in a bioconjugate chain. It should be noticed that the conjugation of met-enkephalin with P(D-ran-O300) clearly influences the sizes of resulting aggregates compared with the neat copolymer. The mesoglobules of bioconjugate have significantly larger sizes than those of the copolymer, regardless of heating procedure or solution concentration (Figure S8). The mesoglobules formed by P(D-ran-O300)-MEDns remained stable for at least three days when kept above TCP of bioconjugate.


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3.4. Coating of Bioconjugate Mesoglobules by Crosslinked Shells Functionalized with Target Agent Mesoglobules of bioconjugate obtained by abrupt heating at fixed concentration of 0.2 g/L, in presence of SDS (s/p = 0.2) were coated with polymeric shell ensuring stability of nanoparticles and protection of met-enkephalin moieties. The polymeric shell on the mesoglobule surface was synthesized in a two-step procedure by the radical polymerization of NIPAM in the presence of the N,N′-bis(acryloyl)cystamine (BAC) cross-linker, which enabled degradation of the shell by glutathione.36 During the second step of coating the surface of nanocarries was functionalized by a targeting moiety - RGD with fluorescent label (CFRGD-KAAm) (Figure 4A).

CFRGD-KAAm

molecules contained an acrylamide function which allowed for their covalent attachment to PNIPAM shell the by copolymerization reaction. After first coating reaction the hydrodynamic radius of particles increased from 15.5 to 43 nm, as shown by DLS measurements (Figure 4B). The shell thickness calculated from the difference in hydrodynamic radii of particles before and after coating was found to be 27.5 nm. The functionalization of nanoparticles with targeting agent was achieved during second coating reaction – a thin shell of 11 nm in thickness containing

CFRGD-KAAm

molecules was

synthesized. The radius of the resulting nanocarriers was about 54 nm.


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Figure 4. A) Schematic illustration of two step coating procedure: Coating 1- formation of first shell layer on mesoglobules; Coating 2 – formation of second shell layer with simultaneous functionalization of the nanoparticle surface with targeting agent. B) DLS size distribution curves before and after each step of coating (measurements were performed at 65°C). C) Fluorescence spectra of bioconjugate mesoglobules before (ME-Nc) and after functionalization with CFRGD-KAAm (Me-Nc-RGD). Fluorescence measurement (Figure 4C) confirmed successful encapsulation of met-enkephalin and introducing of RGD in the structure of nanoparticles. The characteristic emission bands for dansyl-labeled met-enkephalin at the excitation wavelength 323 nm was observed in both spectra, whereas the emission band corresponding to carboxyfluorescein-labeled RGD appeared only after functionalization of the nanoparticles during second coating.


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Based on the fluorescence analyses, it was possible to estimate the concentration of encapsulated MEDns and attached

CFRGD-KAAm

(Figure S9 and S10, Supporting Information)

in the dispersion of nanocarriers. The obtained values for met-enkephalin and RGD were 2.64×10-7 mol/L and 1.01×10-7 mol/L, respectively. Therefore, we can assess that on one encapsulated met-enkephalin molecule, 0.38 targeting units have been attached to the shell. To prove that the RGD targeting agent is accessible for external environment (outside mesoglobule) an enzymatic hydrolysis catalyzed by trypsin was performed for Me-Nc-RGD in buffer solution at 37°C. The digestion products were analyzed by ESI-MS (Figure S11, Supporting Information). Expected digestion product RCF (M = 532.2 g/mol) was found in the mass spectra indicating that hydrolysis occurred. This evidences that the RGD is accessible for big enzyme molecules and that the RGD units are present on the outer sphere of nanoparticles. The stability of ME-Nc-RGD nanoparticles was investigated at room temperature which is below TCP of the bioconjugate and PNIPAM forming nanoparticles shell. As expected, the size of nanoparticles increased at 25°C due to swelling of thermoresponsive core and shell, as previously reported for other core-shell thermoresponsive structures.32,54 Since application of the investigated carrier systems requires their usage in physiological conditions, thermal behavior was also studied at 37°C. Figure 5A shows variations of ܴ୦ଽ଴ upon abrupt heating/cooling cycles from 25 to 37°C. As can be seen at 37°C, nanoparticles revealed a collapsed structure resulting from the shrinkage of PNIPAM shells above its TCP. The thermoresponsiveness of the nanocapsules was fully reversible, even after several cycles, of changing temperature. The mesoglobules serving as cores are spherical. Assuming that the coating process takes place evenly over the mesoglobules surface, spherical nanoparticles should be received. The morphology of nanocarriers was visualized by AFM. The spherical objects clearly visible from


22

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micrographs (Figure 5B) had around 200-250 nm diameter determined from cross-section analysis (Figure 5C). This value is higher than the resulting hydrodynamic sizes from DLS measurements because of the pancake shape of the nanoparticles at the silicon wafer.

Figure 5. A) Radius of ME-Nc-RGD during heating to 37°C and cooling to 25°C and B) AFM picture with C) cross-section analysis of ME-Nc-RGD nanocapsules. AFM samples were prepared at 25°C.


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3.5. Degradation of Nanoparticles It is known that disulfide bonds undergo degradation in the presence of glutathione.55,56 Their presence in crosslinked shell allows for disintegration of nanoparticles. The degradation process was followed by measuring ME-Nc-RGD sizes by DLS after the addition of glutathione to nanoparticles dispersion (Figure 6).

Figure 6. A) Changes in Me-Nc-RGD nanoparticles’ sizes during degradation in the presence of glutathione at 37°C. B) Size distributions of Me-Nc-RGD after certain time of degradation. Complete degradation of nanoparticles occurred within 20 h. The process began from swelling of the particles; their radii constantly increased up to 230 nm within 6 hours and then dramatically decreased (Figure 6A). The nanoparticles degraded into a species of around 1 nm,


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the size characteristic for individual polymer chains dissolved in solution. The size distribution of nanoparticles after certain time of degradation is shown in Figure 6B. Within the degradation process, only one population of particles was observed. 4. CONCLUSION The use of thermoresponsive polymers creates interesting possibilities for obtaining nanocarriers of therapeutic peptides equipped with targeting molecules. Here, we showed the route leading to nanocarriers of met-enkephalin with outer shells containing targeted RGD. The applied synthetic strategy which involved ATRP led to a well-defined thermoresponsive polymer-met-enkephalin conjugate which easily assembles to spherical nanoparticles in water solution upon heating. If proper conditions are chosen (concentration, rate of heating, presence of additives), this process of organization permitted the relatively easy control of nanoparticle sizes. The nanoparticles can be stabilized by coating with crosslinked shells using nucleated radical polymerization of N-isopropylacrylamide. During the coating process, RGD, the targeting agent, equipped with acrylamide bond active in the polymerization process, can be attached to their outer shell. In response to cyclic changes in temperature from below to above the phase transition temperature of the bioconjugate, the nanoparticles were stable and reversibly changed their sizes, which is related to the swelling and collapsing of polymeric chains. Due to the presence of disulfide bonds in the structure of an applied cross-linker shell, nanoparticles undergo complete degradation in the presence of glutathione releasing bioconjugates to the solution. The proposed strategy shows the utilization of thermoresponsive polymers and their properties to construct a new type of anticancer peptide nanocarrier.

ASSOCIATED CONTENT


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Supporting Information. The article is accompanied with supporting information which contains detailed description of peptides synthesis and their modification; characterization of peptides by mass spectrometry and high performance liquid chromatography; gel permeation chromatography analysis, proton nuclear magnetic resonance and particle size distributions of model copolymer; fluorescence calibration curves for peptides, enzymatic hydrolysis of the nanoparticles by trypsin and hydrolysis products characterization by mass spectrometry. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Notes The authors declare no competing financial interest. ACKNOWLEDGMENT The authors acknowledge the National Science Center of Poland (grant NN 209 756740 and NN 209 144136) and a bilateral cooperation project of the Polish and Bulgarian Academies of Sciences for their support. The authors gratefully acknowledge A. Szymura from the School of Pharmacy with the Division of Laboratory Medicine, Medical University of Silesia for preparing the literature study on the bioactivity of met-enkephalin and A. Marcinkowski from Centre of Polymer and Carbon Materials for AFM analyses.


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For Table of Contents Use Only

Smart Polymeric Nanocarriers of Met-enkephalin Roza Szweda,† Barbara Trzebicka,†* Andrzej Dworak,† Lukasz Otulakowski,† Dominik Kosowski,† Justyna Hertlein,† Emi Haladjova,‡ Stanislav Rangelov,‡ and Dawid Szweda† †

Centre of Polymer and Carbon Materials, Polish Academy of Sciences, M. Curie-Sklodowskiej

34, Zabrze, 41-819, Poland ‡

Institute of Polymers, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., bl. 103-A, Sofia,

1113, Bulgaria


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Smart Polymeric Nanocarriers of Met-enkephalin - Biomacromolecules

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