Control of rhGH Release Profile from PEG–PAF Thermogel - American

Apr 7, 2015 - Usha Pramod Shinde, Hyo Jung Moon, Du Young Ko, Bo Kyong Jung, ... The PEG−PAF aqueous solutions underwent heat-induced sol-to-gel...
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Control of rhGH Release Profile from PEG−PAF Thermogel Usha Pramod Shinde, Hyo Jung Moon, Du Young Ko, Bo Kyong Jung, and Byeongmoon Jeong* Department of Chemistry and Nano Science, Ewha Womans University, 52 Ewhayeodae-gil, Seodaemun-gu, Seoul, 120-750, Korea S Supporting Information *

ABSTRACT: Poly(ethylene glycol)-poly(L-alanine-co-L-phenyl alanine) diblock copolymers (PEG−PAF) of 2000−990 Da (P2K) and 5000−2530 Da (P5K) with the different molecular weights of PEGs, but having a similar molecular weight ratio of hydrophobic block to hydrophilic block were synthesized to compare their solution behavior and corresponding protein drug release profiles from their in situ formed thermogels. The PEG−PAF aqueous solutions underwent heat-induced sol-to-gel transition in a concentration range of 18.0−24.0 wt % and 8.0−12.0 wt % for P2K and P5K, respectively. P5K formed bigger micelles than P2K, of a broad distribution, whereas the PAF blocks of P5K developed richer in α-helix than those of P2K in the core of the micelles. As the temperature increased, the micelles underwent dehydration of the PEG, which led to the aggregation of micelles, while the secondary structure of PAF was slightly affected during the sol-to-gel transition. The P5K exhibited higher tendency to aggregate and formed a tighter gel than P2K. Upon injection into the subcutaneous layer of rats, both polymer aqueous solutions formed a biocompatible gel with typical mild inflammatory tissue responses. Recombinant human growth hormone (rhGH) maintained its stability without forming any aggregates in both sol (4 °C) and gel (37 °C) states of the PEG−PAFs. Even though P2K and P5K have a similar molecular weight ratio of hydrophobic block to hydrophilic block, the P5K system exhibited a reduced initial burst release, improved bioavailability, and prolonged therapeutic duration of the rhGH, compared to the P2K system. The current research suggests that a drug release profile is a complex function of self-assembling carriers and incorporated drugs, and thus, a promising protein delivery system could be designed by adjusting the molecular parameters of a thermogel.

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INTRODUCTION Human growth hormone (hGH) is stored in the anterior pituary and is secreted in a pulsatile manner. About 100−2000 μg/day of hGH is secreted from a human, which is strongly age-dependent.1 Recombinant hGH (rhGH) has been used to treat children and adults with growth hormone deficiency (GHD), chronic renal insufficiency, or Turner’s syndrome.2 The minimum effective concentration of rhGH in plasma is 2− 5 ng/mL in order to induce therapeutic concentration of biomarkers such as insulin-like growth factor-I (IGF-I) and insulin-like growth factor binding protein-3 (IGFBP-3).3,4 Due to its short plasma half-life of 3.8−4 h, rhGH should be administered at a daily dose of 26−43 μg/kg either daily or three times per week by subcutaneous injection for children with GHD for several years, which leads to poor patient compliance of the current therapy.5,6 Since it has been reported that a continuous delivery of the rhGH exhibits the same efficacy as the daily injection, rhGH has been a target drug looking for a sustained delivery system.7,8 The first sustained release formulation of rhGH, Nutropin Depot, was launched by Genentech and Alkermes in 1999 in the United States.9,10 Nutropin Depot was a rhGH encapsulated poly(DL-lactide-coglycolide) (PLGA) microsphere system and provided 2 weeks or one month delivery of rhGH by a single injection. However, the system was withdrawn from the market in 2004 due to the cost of the scale-up process, inflammation around the injection site, and denaturation of the rhGH by an acidic microenvironment associated with the degradation of PLGA.11,12 The second © XXXX American Chemical Society

generation of the sustained release system of rhGH, LB03002, was launched in 2007 as a once-per-week injection formulation in Korea by LG Life Sciences.13 LB03002 actually exhibited a three day release profile in vivo, however, the biological consequences of maintaining therapeutic IGF-I level >200 ng/ mL make the system adequate as a once-per-week injection system.14 LB03002 is a rhGH encapsulated hyaluronic acid microparticle system. The microparticles was about 5 μm in size, and was manufactured by the spray drying method. LG Life Sciences completed the clinical trial of LB03002 for idiopathic short stature patients in the United States in 2011 and is in the process of a new drug application (NDA).15 Other than the above systems, sustained release systems for rhGH are still actively being investigated at the research level. The studies focus on decreasing the initial burst release of the drug, prolonging the therapeutic duration, and increasing bioavailability of the drug. Crystallization of rhGH by zinc, followed by complexation of the rhGH crystal with polyarginine or protamine led to polyelectrolyte coated microparticles.16 This system provided an injectable system maintaining 3−4 days of therapeutic levels of rhGH in rats. A nanoparticle system of rhGH encapsulating dextran-g-PLGA was investigated for sustained release of rhGH; 7 day maintenance of the therapeutic levels of rhGH was reported in the mice model.17 Received: October 29, 2014 Revised: April 3, 2015

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DOI: 10.1021/acs.biomac.5b00325 Biomacromolecules XXXX, XXX, XXX−XXX

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Biomacromolecules Table 1. List of Polymers polymer

PEG−PAFa

Mna

Mwb

PDIb

2nd structurec (α/β/r/others)

P2K P5K

EG45−A10.0F1.9 EG112−A28.4F3.5

2000−990 5000−2530

2380 6070

1.2 1.2

34/36/20/10 48/29/22/1

a

Determined by 1H NMR in CF3COOD. bDetermined by GPC using N,N-dimethylformamide as an eluting solvent. Poly(ethylene glycol)s were used as the molecular weight standards. cThe secondary structures of the PAF of PEG−PAFs determined by the amide I band at 1600−1700 cm−1 of the FTIR spectra of the polymer aqueous solutions (in D2O) at 10 °C. P2K (19.0 wt %) and P5K (10.0 wt %) were used. The band positions were assigned as follows: α-helix (α, 1659 cm−1), β-sheet (β, parallel: 1625 and 1632 cm−1, antiparallel: 1696 cm−1), random coil (r, 1646 cm−1), and others, including β-turn (1674 cm−1) and aggregated strands (1613 cm−1).44

conjugated PEG affects the secondary structure of polypeptides as well as the self-assembled nanostructure of the PEGpolypeptide diblock copolymer aqueous solution as reported for PEG-poly(L-alanine), and thus, it is expected to affect the release profile of the incorporated drug.39 The solution behavior of the polymers, biodegradation, and in vitro/in vivo release profiles of rhGH from the in situ formed gels were compared between P2K and P5K to find out molecular clues in designing a protein drug delivery system with a low initial burst release, improved bioavailability, and prolonged therapeutic duration of the incorporated drugs.

Poly(ethylene glycol)-poly(DL-lactide) (PEG−PLA) microspheres were prepared for sustained release of rhGH.18 Compared to the PLGA microsphere system, the PEG−PLA diblock copolymer microspheres exhibited high water content, leading to swelling of the microspheres. Therefore, they improved the diffusion of acids which were generated during the degradation of the microsphere. Another interesting delivery system of rhGH is the hyaluronic acid-conjugated rhGH system. The conjugate system exhibited an excellent transdermal transport property through the receptor mediated process of hyaluronic acid receptor in the skin layer of rats.19 Thermogelling systems that undergo solution-to-gel transition as the temperature increases provide several advantages as a sustained release system of drugs.20−24 The drugs are mixed in a low viscous sol state, followed by injection into a target area, which, due to the thermal energy of the warmblooded animals or human, leads to the formation of a hydrogel depot. The in situ formed hydrogel acts as a sustained release system of the drugs. The aqueous solution of the thermogelling system avoids direct contact between the organic solvent with the protein drug. In addition, the simple sterilization through the microfiltration in a sol state is also a significant advantage over ethylene oxide sterilization of microsphere systems. PLGA, chitosan, polyphosphazene, polycaprolactone, poloxamer derivatives, poly(hydroxypropyl methacrylamide-monolactate and -dilactate), elastin-like polypeptide (ELP)-based polymers, and silk-elastin-like polymers have been reported as biodegradable thermogels.25−33 However, most protein drug delivery systems suffer from a short therapeutic duration and a large initial burst release.34,35 Recently, we have reported on a series of thermogelling polypeptides including polyalanine (PA), poly(alanine-co-phenyl alanine) (PAF), and poly(alanine-co-leucine) (PAL).36−39 The polymers are degraded by the proteolytic enzymes of mammals to amino acids.36 The polypeptide system not only provides the storage stability as an aqueous polymer solution but also exhibits compatibility for biopharmaceuticals such as protein drugs and cells.37,40−42 In particular, recently we reported PEG−PAF thermogel (molecular weight of each block 2000−690; EG45-A6.8F1.4) for sustained release of rhGH with a therapeutic duration over 4 days in rats.40 However, the system also suffered from a large initial burst release of the incorporated drug as well as low bioavailability. In addition, it still needed to prolong the in vivo therapeutic duration with a plasma concentration >2 ng/mL. To solve these problems, in this paper, we are reporting two thermogelling polypeptide systems of PEG−PAFs (P2K and P5K) with increased hydrophobicity than the previous system. They are expected to form a tighter gel and reduce the initial burst release of incorporated drugs, at least. P2K and P5K were prepared from PEGs with molecular weights of 2000 and 5000 Da, respectively, while maintaining a similar molecular weight ratio of hydrophobic block to hydrophilic block. The

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EXPERIMENTAL SECTION

Materials. α-Amino-ω-methoxy-poly(ethylene glycol)s (PEGs; Mn = 1000, 2000, 5000 Da; ID Bio, Korea) were used as received. NCarboxy anhydrides of L-alanine and N-carboxy anhydrides of L-phenyl alanine (KPX Life Science, Korea) were stored at 4 °C in a desiccator filled with drying agents under high vacuum and used as needed. Haematoxylin and eosin Y (H&E) were used as received from SigmaAldrich, U.S.A. Toluene was dried over sodium before use. rhGH (Advanced Protein Technologies Co., Korea) was used as received. Polymer Synthesis. PEG−PAFs were synthesized by ring-opening polymerization of the N-carboxy anhydrides of L-alanine (NCA-Ala) and N-carboxy anhydrides of L-phenyl alanine (NCA-Phe) by using the α-amino-ω-methoxy-PEG as an initiator.36,43 The feed composition was 3.50 g (30.43 mmol) of NCA-Ala/1.10 g (5.76 mmol) of NCA-Phe/5.00 g (2.50 mmol) of α-amino-ω-methoxy-PEG for P2K, and 3.50 g (30.43 mmol) of NCA-Ala/1.10 g (5.76 mmol) of NCAPhe/5.00 g (1.00 mmol) of α-amino-ω-methoxy-PEG for P5K. The polymers were purified by fractional precipitation into diethyl ether, followed by evaporation of the residual solvent under vacuum. The polymers were dialyzed in water using a membrane and freeze-dried. The final yields were 68 and 60% for P2K and P5K, respectively (Table 1). 1 H NMR Spectroscopy. 1H NMR spectra of PEG−PAF in CF3COOD (500 MHz NMR spectrometer; Varian) were used to determine the composition and molecular weight of the polymer. 1H NMR spectral changes of the PEG−PAF aqueous solutions in D2O were investigated as a function of temperature in a temperature range of 10−50 °C. The solution temperature was equilibrated for 15 min at each temperature before measurements. Gel Permeation Chromatography. A gel permeation chromatography (GPC) system consisted of a pump (SP930D; Younglin, Korea) with a refractive index detector (R1750F; Younglin, Korea) and an OHpak SB-803QH column (Shodex, Japan) was used to obtain the molecular weight distribution of the polymers. N,N-Dimethylformamide was used as an eluting solvent. Poly(ethylene glycol)s in a molecular weight range of 400−20 000 Da were used as molecular weight standards. Phase Diagram. The sol−gel transition of the PEG−PAF aqueous solution was investigated by the test tube inverting method. The aqueous polymer solution (1.0 mL) was put in the test tube with an inner diameter of 11 mm. The transition temperatures were determined by a flow (sol)−no flow (gel) criterion with a temperature increment of 1 °C per step. Each data point is an average of three measurements. B

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mg) was injected into a vial with a diameter of 1.1 cm at 37 °C. After confirming gel formation by incubating the aqueous polymer solution for 2 min at 37 °C, the release medium (phosphate buffered saline at pH = 7.4, 0.02% Tween 20, 0.02% sodium azide, 2.0 mL) at 37 °C was added on the top of the gel. Triplicate experiments (n = 3) were performed. The vials were kept in a shaking incubator (VS-101Si, Vision Scientific Co., Korea) at 50 rpm. The release medium was replaced at the designated time interval. The amount of rhGH released was analyzed by HPLC (Waters 1525B, U.S.A.) with photodiode detector (Waters 2998, U.S.A.) at a wavelength of 214 nm. Acetonitrile/water cosolvent was used as an eluting solvent in a gradient manner. For in vivo studies, the rhGH formulation (0.5 mL, rhGH dose = 5.0 mg/kg rat) was injected into the subcutaneous layer of rats (body weight 200−230 g) and blood samples (200 μL) were taken from the tail veins of the rats at designated time interval. The blood samples were allowed to clot for 30 min in Eppendorf tubes, and then centrifuged for 15 min at 1000g according to the protocol of the Elisa R&D system. And the aliquots were stored at −20 °C. The serum drug concentration was assayed by the ELISA assay kit (R&D System Quantikine, U.S.A.). Animal Procedure. All experimental procedures using animals were in accordance with the Institutional Animal Care and Use Committees (IACUC) guidelines and were approved by the Committee of Ewha Womans University (approval code: IACUC No: 2013−01−081).

Dynamic Mechanical Analysis. Changes in modulus of the PEG−PAF aqueous solutions were investigated by dynamic mechanical analyzer (Thermo Haake, Rheometer RS 1) as a function of temperature in a range of 10−50 °C. The aqueous polymer solution was placed between parallel plates of 25 mm diameter with a gap of 0.5 mm. To minimize water evaporation during the experiment, the plates were enclosed in a water saturated chamber. The data were collected under controlled stress (4.0 dyn/cm2) and at a frequency of 1.0 rad/s. The heating rate was 1.0 °C/min. Transmission Electron Microscopy (TEM). The PEG−PAF aqueous solution (10 μL; 0.01 wt %) at 10 °C was placed on the carbon grid and excess solution was blotted with filter paper. The grids were dried at room temperature for 24 h. The microscopy images of the polymer assemblies were obtained by a JEM-2100F (JEOL, Japan) with an accelerating voltage of 200 kV. Dynamic Light Scattering. The apparent size of a polymer or polymer aggregates in water (0.10 wt %) was studied by a dynamic light scattering instrument (ALV 5000−60 × 0) as a function of temperature in a range of 10−50 °C. A YAG DPSS-200 laser (Langen, Germany) operating at 532 nm was used as a light source. The results of dynamic light scattering were analyzed by the regularized CONTIN method. Circular Dichroism (CD) Spectroscopy. CD spectra of the PEG−PAF aqueous solution were measured by a CD instrument (J810, JASCO, Japan) as a function of concentration in a range of 0.001−0.10 wt % at 10 °C. In addition, the mean residue ellipticity of the PEG−PAF aqueous solution was obtained as a function of temperature in a range of 10−50 °C at a fixed concentration of 0.005 wt % to study the change in secondary structure of the polypeptide as a function of temperature. FTIR Spectroscopy. The IR spectra (FTIR spectrophotometer FTS-800; Varian, U.S.A.) of the PEG−PAF aqueous solutions in D2O were investigated as a function of temperature in a range of 10−50 °C to study the secondary structural changes of the polypeptide as a function of temperature. The resolution of the FTIR spectra was 1 cm−1. To analyze the secondary structure of the polypeptide, deconvolution of the FTIR spectra was done in the amide I band region.44 The deconvoluted spectra were fitted with a Gaussian− Lorentzian sum function using XPSPEAK software 4.1 (RCSMS Lab). In Vivo Degradation of PEG−PAF Gels. The PEG−PAF aqueous solutions (0.5 mL, P2K; 19.0 wt %, P5K; 10.0 wt %) were injected into the subcutaneous layer of rats (body weight: 200−230 g). The remaining gels at 7 days after the implantation were taken, and the molecular weights of the recovered polymers were analyzed by the GPC system. The in vitro degradation of PEG−PAFs was studied at the polymer concentration of 1.0 wt % in phosphate buffered saline (150 mM) at pH = 7.4 for the control experiment. The samples were analyzed by the GPC system. Histocompatibility Study. The rats were sacrificed 7 days after implantation of the gel in the subcutaneous layer. Neutral buffered formalin (NBF) solution was prepared by mixing formaldehyde solution (37−40%; 100 mL), sodium phosphate monobasic (4.0 g), sodium phosphate dibasic (6.5 g), and deionized water (900 mL). The tissue surrounding the gel was stored in NBF solution at room temperature. The specimens were dehydrated in a graded series of ethanol, and the tissues were embedded in paraffin. The microtomed sections of the tissue with a thickness of about 6 μm were stained with H&E and examined by microscopy (Olympus lX71-F22PM, Japan). Stability of rhGH in PEG−PAF Systems. rhGH formulations in aqueous solutions (1.0 mg/0.5 mL) of three different PEG−PAFs (P2K, 19.0 wt %; and P5K, 10.0 wt %) were stored at 4 °C (sol state) and 37 °C (gel state) for 7 days. After collecting the samples for 0 to 7 days, rhGH was analyzed by using the HPLC system (Waters 1525B, U.S.A.) with photodiode detector (Waters 2998, U.S.A.) at a wavelength of 214 nm. The acetonitrile/water cosolvent system was used as a mobile phase in a gradient manner, and a Proteinca C4, 214ATS54 (Vydac), U.S.A.; 5 μm, 4.6 mm × 250 mm) analytical column was employed with a flow rate of 1.0 mL/min. Sustained Release of rhGH. The PEG−PAF aqueous solution (0.5 mL; P2K; 19.0 wt %, P5K; 10.0 wt %) containing the rhGH (1.0

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RESULTS AND DISCUSSION PEG−PAF was synthesized by ring opening copolymerization of N-carboxy anhydrides of L-alanine and N-carboxy anhydrides of L-phenyl alanine by using α-amino-ω-methoxy PEG.36,43 PEG with molecular weights of 2000 and 5000 Da were used as the initiators for P2K and P5K, respectively, and the molecular weights of the PEG−PAFs were controlled for the polymer aqueous solutions to exhibit thermal gelation in a physiologically important temperature range of 10−40 °C. The areas of the 1H NMR spectra at 1.2−1.9 ppm (−CH3 of PAF), 3.8−4.2 ppm (−CH2CH2O− of PEG), and 7.0−7.5 ppm (phenyl group of PAF) were used to calculate the composition and the molecular weight of the PEG−PAF (Figure 1). In addition, the

Figure 1. 1H NMR spectra of the PEG−PAFs (CF3COOD). The peaks at 1.2−1.9 ppm (−CH3 of PAF), 3.8−4.2 ppm (−CH2CH2O− of PEG), and 7.0−7.5 ppm (phenyl group of PAF) were used to calculate the molecular weight of the PEG−PAF.

molecular weight distribution of the PEG−PAFs was determined by GPC by using N,N-dimethylformamide as an eluting solvent. The unimodal distribution of the polymer molecular weights in GPC chromatograms, as will be shown in the degradation section of the PEG−PAF in this paper, suggested that the PEG−PAFs were well-prepared. The molecular weights and molecular weight distribution of the C

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for alanine and phenyl alanine, respectively, indicating a significantly greater hydrophobicity of phenyl alanine over alanine.49 As will be discussed, P5K is richer in α-helical content more than P2K and forms micelles with a larger size in water. Even though P2K (2000−990) and P5K (5000−2530) have a similar molecular weight ratio of hydrophobic block to hydrophilic block, P2K aqueous solutions exhibited sol-to-gel transition in a concentration range of 18.0−24.0 wt %, whereas P5K aqueous solutions exhibited sol-to-gel transition in a lower concentration range of 8.0−12.0 wt %. At concentrations lower than the sol-to-gel transition observed, the modulus of the polymer aqueous solution was not large enough to resist the flow when the test tube was inverted, and thus they were regarded as a sol. At polymer concentrations greater than the sol-to-gel transition observed, the aqueous polymer solution formed a gel in the investigated temperature range of 0−60 °C. Considering practical application as an injectable drug delivery system, we are interested in polymer aqueous solutions with the sol-to-gel transition temperature of 20−35 °C. Therefore, 19.0 and 10.0 wt % were selected for P2K and P5K aqueous solutions, respectively. The dynamic mechanical analysis of the polymer aqueous solution showed sharp increases in both the storage modulus (G′) and loss modulus (G″) of P2K and P5K as the temperature increased (Figure 2b). G′ and G″ are measures of the elastic component and viscous component of the complex modulus (G*), respectively. The crossing point of G′ over G″ has been considered as a criterion for sol-to-gel transition temperature.50,51 The crossing point of P2K (19.0 wt %) and P5K (10.0 wt %) aqueous solutions were 31 and 27 °C, respectively. The gel moduli (G′) at 37 °C were 135 and 240 Pa for P2K and P5K, respectively. The modulus of a gel is a measure of messy size which, therefore, affects the diffusion rate of the incorporated drug.52,53 The greater gel modulus of P5K at 37 °C, compared with P2K might also contribute to the slow release rate of the drug, as will be discussed. The photo images of the sol and gel states of the current PEG−PAF (P5K) are also provided (Figure 2c). The PEG−PAF consists of a hydrophilic PEG block and a hydrophobic PAF block; therefore, the polymers self-assemble in water to form core−shell nanostructures. TEM images developed from the polymer aqueous solution (0.01 wt %) showed spherical micelle structures (Figure 3). The P5K

PEG−PAFs are summarized in Table 1. The diblock copolymers were assigned as P2K and P5K for the PEG− PAFs with the molecular weight of 2000−990 and 5000−2530, respectively. Aqueous solutions of P2K and P5K undergo sol-to-gel transition as the temperature increases. The sol−gel phase diagrams of PEG−PAF aqueous solutions were determined by the test tube inverting method. Based on the flow (sol) and no flow (gel) criterion, the sol-to-gel transition temperatures of the PEG−PAF aqueous solutions were determined when the vials were inverted with a temperature increment of 1 °C/step. Because the sol-to-gel transition accompanied more than a 1000× increase in modulus, the visual observation by the above method consistently determined the transition temperatures within a precision of ±2 °C (Figure 2a). The concentration

Figure 2. (a) Phase diagram of the PEG−PAF aqueous solutions determined by the test tube inverting method (n = 3). (b) Storage modulus (G′) and loss modulus (G″) of the P2K (19.0 wt %) and P5K (10.0 wt %) aqueous solutions as a function of temperature. (c) Photos of sol (4 °C) and gel (37 °C) states of the P5K aqueous solution.

range exhibiting sol-to-gel transition and sol-to-gel transition temperatures depend on the balance between hydrophobicity and hydrophilicity of polymers. As the hydrophobicity of the polymer increases, the transition temperature and the concentration range that the sol-to-gel transition being observed tend to decrease.23,45,46 However, in the case of polypeptides, the secondary structure of polypeptides also affects the sol-to-gel transition and the trend can not be predicted by a simple hydrophobicity scale, as reported in thermogelling poly(L-alanine) and poly(DL-alanine).47,48 Based on the partition coefficient of amino acid residues between 1octanol and water, the hydrophobicity was defined by ΔG = −RT ln K, K = [X]octanol/[X]water. ΔGs were −0.39 and −2.27

Figure 3. TEM images of P2K and P5K developed from their aqueous solutions at 0.01 wt %. The scale bar is 200 nm.

formed a broad distribution of micelles bigger than the P2K. Analyses of TEM images showed that P2K and P5K micelles exhibited average diameters of 30 (±8) nm and 52 (±15) nm, respectively. Self-assemblies of the polymers were also investigated by dynamic light scattering of the polymer aqueous solution (0.10 D

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Biomacromolecules wt %) as a function of temperature. The study at a low concentration region of 0.10 wt % can provide the information on intrinsic properties of the self-assemblies of the P2K and P5K. At 10 °C, peak average micelle size of the P2K and P5K were 37 and 105 nm, which increased to 106 and 190 nm, respectively, as the temperature increased to 50 °C (Figure 4).

Figure 5. Circular dichroism spectra of PEG−PAF aqueous solutions as a function of concentration at a fixed temperature of 10 °C. The legends are concentrations in wt % in water.

Figure 4. Size distribution of micelles determined by dynamic light scattering of the polymer aqueous solution (0.10 wt %) as a function of temperature.

not significantly changed in a temperature range of 10−50 °C, suggesting that the PEG−PAFs maintained their secondary structure in this temperature range. FTIR spectra of P2K and P5K exhibited characteristic amide I bands at 1625 and 1655 cm−1, which are known as characteristic bands of β-sheet and α-helix, respectively. For the semiquantitative analyses of the secondary structures of the P2K and P5K, the amide I bands (1600−1700 cm−1) of PEG− PAF aqueous solutions (P2K, 19.0 wt %; P5K, 10.0 wt % in D2O) at 10 °C were deconvoluted (Supporting Information, Figure S2). The analysis of the secondary structure of polypeptide by the curve fitting program on the amide I band of FTIR has limitations due to the fact that the exact quantity can be affected by the standard peak position. Therefore, it provides semiquantitative information. However, the FTIR spectra surely suggest that α-helical content is richer in P5K than in P2K, and β-sheet content is richer in P2K than in P5K (Table 1). The amide I bands of P2K and P5K were investigated as a function of temperature (Figure 6). Basically, the characteristic amide I band related to the secondary structure of the polypeptide maintained in a temperature range of 10−50 °C. Both the FTIR and the CD spectra of P2K and P5K suggested that the secondary structures of the PEG−PAFs were slightly affected in a temperature range of 10−50 °C. The secondary structure of the polypeptide depends on the molecular weight of the conjugated PEG to the polypeptide. As the molecular weight of PEG of PEG-poly(L-alanine) diblock copolymers increased, β-sheet content of the poly(Lalanine) decreased, and the α-helix content of the poly(Lalanine) increased.39,59 The current PEG−PAF exhibited a similar trend. The P2K with a small molecular weight PEG formed a comparable composition of α-helix and β-sheet secondary structures, whereas the P5K with a large molecular weight PEG dominantly formed a α-helix secondary structure. The P5K may experience the steric hindrance of PEG in

Similar to TEM images, P5K developed a bigger micelles and broader distribution of the micelle size than P2K. Due to the fact that micelles could be deformed during the water evaporation procedure for TEM experiments, the exact size of micelles observed in TEM and dynamic light scattering studies might be different; however, the trend in micelle size distribution was similar. The fact that sol-to-gel transition temperature of P5K is lower than P2K and micelles of P5K is bigger than P2K suggested that the aggregation of P5K micelles could be more extensive than P2K, which might contribute to the greater modulus of the P5K gel than P2K gel. CD and FTIR spectra provide information on the secondary structures of the polypeptides. While the CD spectra provide the secondary structure information on the polypeptides at low concentrations, FTIR spectra provide the secondary structure information on the polypeptides at high concentrations.38,44,54 CD spectra of P2K and P5K aqueous solutions were investigated as a function of concentration. Above the 0.05 wt %, CD spectra of P2K and P5K showed a red shift to 225 nm and characteristics of the secondary structure of the polypeptides were lost in the CD spectra due to the micelle formation (Figure 5). This phenomenon is well-known for amphiphilic polypeptides.55−57 The critical micelle concentration (cmc) of P2K and P5K can be estimated as 0.01−0.05 wt % by the CD spectra. The cmc determined by CD spectra is reported to be similar to the cmc determined by the UV− visible or fluorescence spectroscopy.47,58 Therefore, CD spectra of P2K and P5K were investigated at 0.005 wt % to study changes in the secondary structure of PAF as a function of temperature (Supporting Information, Figure S1). CD spectra of P5K exhibited a typical α-helix with a maximum at 195 nm and two minima at 208 and 220 nm. P2K exhibited both α-helix and β-sheet secondary structures. The CD spectra of P2K and P5K slightly decreased their magnitude as the temperature increased from 10 to 50 °C. However, the overall shapes were E

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increased, suggesting that the dehydration of hydrophilic PEG block and decrease in molecular motion of the both the PEG and PAF blocks are the major driving factors in the sol-to-gel transition of PEG−PAF, similar to previously reported thermogelling systems of PEG/PLGA, polyphosphazene, and PEG/polycaprolactone.25,27,60−62 Even though similar trends were observed for P2K and P5K aqueous solutions, the PEG peak of P5K was most seriously collapsed at above 30 °C, indicating the slower molecular motion and the more significant dehydration of P5K than P2K in the gel state. This observation could also be related to the higher modulus of the P5K gel than the P2K gel at 37 °C. Based on the DLS, CD, FTIR, and 1H NMR studies of the PEG−PAF aqueous solutions as a function of temperature, the sol-to-gel mechanism of P2K and P5K could be compared as follows. Both P2K and P5K consisting of hydrophilic PEG block and hydrophobic PAF block form micelles in water. P5K with a bigger PEG block develops micelles with a greater shell than P2K, whereas the PAF blocks of P5K develop richer in αhelix than those of P2K in the core of the micelles. The micelles undergo dehydration of the PEG, which lead to the aggregation of micelles, while the secondary structure of PAF are slightly affected during the sol-to-gel transition. The P5K have higher tendency to aggregate and form a tighter gel than P2K. To confirm the gel formation in vivo, the polymer aqueous solution (0.5 mL/rat, P2K; 19.0 wt %, and P5K; 10.0 wt %) was injected into the subcutaneous layer of rats. Photos around the in situ formed gel were taken 7 days after the injection (Figure 8a). The tissue compatibility of the in situ formed gel was

Figure 6. FTIR spectra of P2K (19.0 wt %) and P5K (10.0 wt %) aqueous solutions as a function of temperature. Amide I band region at 1600−1700 cm−1 is shown.

developing the β-sheet structure of the polypeptide which requires the close alignments of the PAFs. The 1H NMR spectra (D2O) of P2K (19.0 wt %) and P5K (10.0 wt %) aqueous solutions were investigated as a function of temperature (Figure 7). As the temperature increased, the ethylene peak of PEG centered at 3.40 ppm downfield-shifted to 3.80 ppm, and the methyl peak of PAF centered at 1.15 ppm downfield-shifted to 1.50 ppm. Both PEG and PAF peaks were broadened and collapsed. In addition, the water (HDO) peak centered at 4.7 ppm was broadened as the temperature

Figure 8. (a) In vivo gel formation by subcutaneous injection of PEG−PAF aqueous solutions (P2K; 19.0 wt % and P5K; 10.0 wt %) into rats. The photos around the implanted site were taken 7 days after the subcutaneous injection. The area inside the yellow-dotted curve indicates the gel region. The centimeter ruler is also shown in the photos. (b) H&E stained images around the implant 7 days after subcutaneous injection of PEG−PAF aqueous solutions (P2K; 19.0 wt % and P5K; 10.0 wt %) into rats. The scale bar is 100 μm. The bluedotted curve is the boundary between the gel and surrounding tissue.

investigated for the tissues around the implant 7 days after the subcutaneous injection. Microscopic images of the tissues around the implanted hydrogel were obtained after staining by H&E agents. The images showed the histocytes (dark purple) and blood cells (red) migrated into the hydrogel, which are typical mild tissue responses of a biocompatible hydrogel implanted in the subcutaneous layer of rats (Figure 8b).63,64

Figure 7. 1H NMR spectra of P2K (19.0 wt %) and P5K (10.0 wt %) aqueous solutions (D2O) as a function of temperature. F

DOI: 10.1021/acs.biomac.5b00325 Biomacromolecules XXXX, XXX, XXX−XXX

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Biomacromolecules

and P5K, respectively (Figure 10a). The smaller initial burst release of rhGH from P5K indicated the favorable interactions

Degradation of the PEG−PAF implanted in the subcutaneous layer of rats was analyzed by the GPC system (Figure 9).

Figure 9. Degradation of PEG−PAFs. GPC traces of PEG−PAFs (P2K and P5K) after incubation for 7 days in the subcutaneous layer of rats in vivo (gray dashed curve), the polymers stored in PBS at pH = 7.4 (black solid curve), and raw polymers (black thick solid curve) were compared.

The retention time at 13.7 min (P2K) and 15.4 min (P5K) of the raw polymers (thick black solid curves) moved to 15.2/17.7 min (P2K) and 15.9/17.6 min (P5K), respectively, due to the degradation of polymers (gray dashed curves) in the subcutaneous layer of the rats. Previously, we reported that PEG−PAF can be degraded by cathepsin B, cathepsin C, and elastases, which are proteolytic enzymes in the subcutaneous layer of mammals.36 The gel prepared from P2K was more significantly degraded than P5K under in vivo conditions. Assuming similar hydrophobicity of the P2K and P5K, the higher molecular weight of PEG of P5K might interfere against the enzymatic degradation of the polymers more effectively than smaller molecular weight of P2K. In addition, the modulus of P5K gel was greater than P2K, indicating smaller messy size of the P5K gel; thus, penetration of enzymes into the P5K gel might be more interfered than P2K.52,53 On the other hand, the GPC chromatograms of P2K and P5K incubated in phosphate buffered saline (gray solid curves) were similar to those of the raw polymers, indicating the stability of both polymers in the phosphate buffered saline. Thermogelling P2K (19.0 wt %) and P5K (10.0 wt %) systems were investigated for the sustained release of rhGH. First, the stability of rhGH was studied in the PEG−PAF aqueous solution in a sol state (4 °C) as well as in a gel state (37 °C) after incubation for 0, 1, 3, 5, and 7 days. The retention time of rhGH was consistently observed for both P2K and P5K formulation recovered from the sol and gel states without other significant peaks coming from aggregation or denaturation of the rhGH (Supporting Information, Figure S3). The aggregated or denatured rhGH might induce immunogenic responses as in the case of the PLGA system.65 This study suggests that rhGH is stable in the PEG−PAF formulation in a sol state as well as in a gel state. In vitro release studies of rhGH from the in situ formed P2K and P5K gels were carried out to compare the initial burst release of the drug and the diffusion-controlled drug release. The rhGH was loaded with 1.0 mg/0.5 mL formulation. The transition temperature was slightly (2 ng/ mL) of rHGH in rats even though ionic interactions between the carrier and rhGH were introduced by using intentionally incorporated protamine and amino groups of the polymers.35,67,68 Previous PEG−PAF (molecular weight of each block 2000−690; EG45−A6.8F1.4) also exhibited low bioavailability and a sustained release of hGH for 4 days.40 P5K formulation significantly improved bioavailability of rhGH and provided 6 days of a therapeutic level of rhGH in rats without any other chemical modifications. As proven the stability of the rhGH in the gel state of PEG−PAF, the current polypeptide thermogel provides specific advantages for the rhGH delivery over the previous PLGA based microsphere system. Due to its high molecular weight of 67000 Da and amphiphilic nature, rhGH can be partitioned in the hydrophobic core consisting of hydrophobic PAF through hydrophobic interactions between rhGH and PAF as well as in the hydrophilic interstitials consisting of hydrophilic PEG and water. The release profile of the rhGH from the thermogel is governed by a complex function of the concentration, hydrophobicity, aggregation of the polymer, interactions between polymers and rhGH, and degradation of the polymer. rhGH has a α-helical secondary structure, and might entangle with polymers, where larger molecular weight polymers with α-helical secondary structure (P5K) can hold the protein drug more effectively than the smaller molecular weight polymer with β-sheet structure (P2K). Slower degradation of P5K than P2K might contribute the longer therapeutic duration of the P5K system than the P2K system. Current P5K provide an excellent bioavailability of rhGH and therapeutic duration over 6 days without a significant initial burst release, which results from the favorable combination of these variables.

Article

ASSOCIATED CONTENT

S Supporting Information *

CD spectra of PEG−PAFs as a function of temperature, deconvolution of amide I bands (FTIR spectra) of PEG−PAF aqueous solutions (P2K; 19.0 wt %, and P5K; 10.0 wt % systems in D2O) at 10 °C, stability of the rhGH in PEG−PAFs in sol and gel states of P2K and P5K. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: +82 2 3277 2384. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Research Foundation of Korea Grant funded by the Korean Government (MSIP; Grant Number: NRF-2012M3A9C6049835). D.Y.K. is thankful to the Solvay Scholarship.

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REFERENCES

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CONCLUSIONS PEG−PAFs with different PEG molecular weights, but a similar molecular weight ratio of PAF block to PEG block were synthesized, and their aqueous solution behavior and release profiles of rhGH form their in situ formed thermogels were compared. Self-assemblies of the polymers were investigated by TEM and dynamic light scattering. The conformational changes in PAF and PEG blocks were studied by using CD, FTIR, and 1 H NMR spectroscopy as a function of temperature. P2K and P5K formed micelles with a different size distribution, where the core of micelles also exhibited a different secondary structure. The sustained release of rhGH for up to 6 days with a small initial burst release of the rhGH were realized by using P5K, suggesting that the system is very promising as a sustained release system of the protein drug. The increased hydrophobic interactions between rhGH and high molecular weight PAFs, the decreased molecular motion of PEG in the gel state, and the difference in micelle aggregation might contribute to such a release behavior of rhGH from the P5K thermogel. This paper suggests that a promising protein drug release profile could be accomplished through controlling molecular parameters of the PEG−PAF thermogel. H

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