Robust Polyion Complex Vesicles (PICsomes) under Physiological

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Robust polyion complex vesicles (PICsomes) under physiological condition reinforced by multiple hydrogen bond formation derived by guanidinium groups Mao Hori, Horacio Cabral, Kazuko Toh, Akihiro Kishimura, and Kazunori Kataoka Biomacromolecules, Just Accepted Manuscript • Publication Date (Web): 29 Aug 2018 Downloaded from http://pubs.acs.org on August 29, 2018

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Robust polyion complex vesicles (PICsomes) under physiological condition reinforced by multiple hydrogen bond formation derived by guanidinium groups Mao Hori, † Horacio Cabral, † Kazuko Toh,∥ Akihiro Kishimura, ‡,§ Kazunori Kataoka ∥,⊥,*

†Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, 73-1 Hongo, Bunkyo-ku, Tokyo, 113-8656, Japan ‡Department of Applied Chemistry, Faculty of Engineering, Kyushu University, 744 Moto-oka, Nishi-ku, Fukuoka 819-0395, Japan §Center for Molecular Systems, Kyushu University, 744 Moto-oka, Nishi-ku, Fukuoka 8190395, Japan ∥Innovation Center of NanoMedicine (iCONM), Kawasaki Institute of Industrial Promotion, 325-14 Tonomachi, Kawasaki-ku, Kawasaki, 210-0821, Japan ⊥Policy Alternatives Research Institute, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

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KEYWORDS Polyion complex; Block copolymer; Polymersome; Guanidium group; Hydrogen bonding; Nanocarrier

ABSTRACT

Polyion complex vesicles (PICsomes) formed from a self-assembly of an oppositely charged pair of block- and homo-polyelectrolytes have shown exceptional features for functional loading of bioactive agents. Nevertheless, the stability of PICsomes is often jeopardized in physiological environment, and only PICsomes having chemically crosslinked membranes have endured in harsh in vivo conditions, such as in blood stream. Herein, we developed versatile PICsomes aimed to last in in vivo settings by stabilizing their membrane through a combination of ionic and hydrogen bonding, which is widely found in natural proteins as salt bridge, by controlled introduction of guanidinium groups in the polycation fraction toward concurrent polyion complexation and hydrogen bonding. The guanidinylated PICsomes were successfully assembled in physiological salt condition, with precise control of their morphology by tuning the guanidinium content, and the ratio of anionic and cationic components. Guanidinylated PICsomes with 100 nm diameter, which are relevant to nanocarrier development, were stable in high urea concentration, physiological temperature, and under serum incubation, persisting in blood circulation in vivo.


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1. Introduction Supramolecular assemblies formed by well-designed synthetic polymers are attracting much attention as versatile nanocarrier platforms with precisely controlled architectures and functionalities on demand. Among them, nanostructured assemblies based on the interaction between a pair of oppositely charged macromolecules, i.e. polyion complexes (PICs), present remarkable features for working with biomacromolecules, such as the possibility to prepare them in aqueous media without any organic solvent and their ability to integrate a variety of charged compounds, including nucleic acids and proteins1-5. We have recently developed submicronsized vesicular PICs (PICsomes) as resourceful nano-assemblies through the specific combination of a pair of oppositely-charged polyelectrolytes, at least one of which is a block copolymer with non-charged or charge-neutralized hydrophilic segment, such a pair as the block copolymer of polyethylene glycol (PEG) with α,β-polyaspartic acid, PEG-b-poly(α,β-aspartic acid) (PEG-PAsp), and the cationic polyaspartamide derivative, poly([5-aminopentyl]-α,βaspartamide) (P(Asp-AP))6. One of the most unique characteristics of PICsomes is that they can be prepared spontaneously just by simply mixing the polymeric components, exhibiting narrow size distribution without any further purification procedures7. Moreover, by taking advantage of the inner cavity and semipermeable membrane structure of PICsomes, we recently confirmed their capability as enzymeloaded nanoreactors working in vivo toward the development of novel enzyme-based therapeutics8, 9. However, a major issue of PICsomes in biomedical application still needs to be solved: They are sensitive to ionic strength and are difficult to maintain their original vesicular


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structure under physiological conditions10, 11. Thus, for biomedical purposes, the structural stabilization of PICsomes is indispensable12. In previous studies, we have applied chemical crosslinking for stabilizing the PICsomes6, yet this procedure may hamper the integrity of the compounds loaded in the inner aqueous cavity, and requires prolonged reaction time followed by repetitive and complicated purification steps. In addition, the stabilization of PICsomes by covalent crosslinking may cause impaired clearance from the body, which could limit their usage as in vivo nanoreactors and drug nanocarriers. Alternatively, developing straightforward PICsome formulations capable of concomitant spontaneous processes of assembly and stabilization based on weak intermolecular forces may overcome the abovementioned issues, and provide the necessary versatility and robustness for developing functional systems working in vivo. Herein, we focused on reinforcing the PICsomes by introducing multivalent intermolecular hydrogen bonds, which are simple in the formation step, i.e. spontaneously forming just by mixing, and can be tailored, for adapting the nanostructures to the surroundings13-15, into the PIC membrane toward the development of versatile PICsome platforms enduring the harsh in vivo environments. As a hydrogen donor, we adopted the guanidinium group, which is found in natural proteins and can work also as a cation source (pKa = 12.5-13.8) to interact with carboxyl groups of PEG-PAsp in PICsomes. Thus, by controlled introduction of guanidinium group into the side chain of the cationic component (P(Asp-AP)), we successfully assembled PICsomes with unilamellar structure and distinct size distribution that maintained their form in physiological salt concentration, even in the presence of 10% serum. The ability of guanidinylated PICsomes in the 100-nm scale (Nano-PICsomes) to stand the in vivo environments was further confirmed in the bloodstream of mice by intravital confocal


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microscopic imaging. Thus, our proposed stabilization strategy could be effectively applied to PICsomes, as well as other PIC-based nanostructures, for extending their operation in vivo.

2. Materials and Methods 2.1. Materials β-Benzyl L-aspartate N-carboxy anhydride (BLA-NCA) was purchased from Chuo Kaseihin Co., Inc. (Tokyo, Japan). α-Methoxy-ω-propylamino-poly(ethylene glycol) (mPEG-NH2) (Mw = 2,200; Mw/Mn = 1.05) was bought from NOF Co. Ltd. (Tokyo, Japan), and purified through an ion exchange column, CM sephadex C-50 (GE Healthcare UK Ltd.; Buckinghamshire, UK). nButylamine and 1,5-diaminopentane (DAP) were purchased from Tokyo Chemical Industry Co. Ltd. (Tokyo, Japan) and distilled over CaH2 under normal and reduced pressure, respectively. N,N-Dimethylformamide (DMF), dichloromethane (CH2Cl2), N-methyl-2-pyrrolidone (NMP), and benzene were purchased from Nacalai Tesque, Inc. (Kyoto, Japan). DMF, CH2Cl2, and NMP were distilled before use. n-Hexane and ethyl acetate were purchased from GODO Co., Ltd. (Tokyo, Japan). 3,5-Dimethyl-1-guanylpyrazole nitrate (DMGP) was purchased from SigmaAldrich Co. (St. Louis, MO, USA). Sodium carbonate, guanidine hydrochloride, sodium chloride, deuterated dimethyl sulfoxide (DMSO-d6), and deuterium oxide (D2O) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Cy3-NHS ester mono-reactive dye pack was purchased from GE Healthcare UK, Ltd. 1-Ethyl-3-(3-(dimethylamino)propyl) carbodiimide hydrochloride (EDC) was purchased from Wako Pure Chemical Industries, Ltd. Balb/c mice (female, 5 weeks old) were purchased from Charles Liver Laboratories Japan, Inc. (Kanagawa, Japan).


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2.2. Methods 2.2.1. 1H-NMR 1

H-NMR spectra were measured with a JNM-ECS 400 (JEOL; Tokyo, Japan) at 400 MHz in d6-

DMSO (measurement temperature = 80 ºC) or in D2O (measurement temperature = 25 ºC) according to the sample solubility.

2.2.2. Size exclusion chromatography (SEC) SEC measurements in organic phase were performed using a TOSOH HLC-8220 system equipped with TSK gel columns (SuperAW4000 and SuperAW3000 × 2; eluent: NMP including 50 mM LiBr; flow rate: 0.3 mL/min; temperature: 40 ºC). SEC measurements in aqueous phase were conducted at room temperature with an HPLC system (JASCO International Co., Ltd; Tokyo, Japan) equipped with a Superdex 200 10/300 GL column (GE Healthcare UK Ltd.; Buckinghamshire, U.K.) and an internal ultraviolet (UV) detector (UV-1575, JASCO, Japan).

2.2.3. Dynamic light scattering (DLS) Size and distribution of polyion complex (PIC) samples were evaluated by dynamic light scattering (DLS) (Zetasizer Nano-ZS, Malvern Instruments; Malvern, U.K.) equipped with a He-


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Ne ion laser (λ = 633 nm). A scattering angle was set to 173º through all the measurements. By using the cumulant method, the intensity-averaged hydrodynamic diameter and polydispersity index (PDI) were calculated.

2.2.4. Transmission electron microscopy (TEM) All the PIC samples were crosslinked before TEM observation according to the previously reported method in order to fix their structure6. Briefly, 10-fold equivalent of EDC (10 mg/mL) to carboxyl groups in PIC sample was added to the PIC solution and gently mixed by pipetting. After 12 h of incubation at room temperature, solvent was exchanged with deionized water by ultrafiltration using Vivaspin® 6 centrifugal concentrator (MWCO = 100 kDa; GE Healthcare UK Ltd). TEM observation was conducted using a JEM-1400 (JEOL, Tokyo, Japan) operated at 150 kV acceleration voltage. 400-mesh copper grids with carbon-coated supporting film (JEOL, Tokyo, Japan) were glow-discharged for 6-8 sec with an Eiko IB-3 ion coater (Eiko Engineering Co. Ltd., Tokyo, Japan). Samples (2 μL) were put on the copper grids, and stained with the same volume of uranyl acetate (2% w/v) for 45-60 sec. The excess amount of solution was removed with a filter paper, and the grids were dried in air at room temperature.

2.2.5. Fluorescence correlation spectroscopy (FCS) measurement FCS analysis was conducted to measure the size distribution of PICsome in the presence of 10% serum protein using a ConfoCor3 module of LSM510 (Carl Zeiss, Oberlochen, Germany) equipped with a Zeiss C-Apochromat 40× water objective. A He-Ne laser (λ = 543 nm) was


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used for excitating of Cy3 dye in PICsome sample prepared from Cy3-labeled PEG-PAsp and P(Asp-AP)6. A 560-615 nm band-pass filter was selected to filter emission. The measured autocorrelation curves were fitted with the Zeiss ConfoCor3 software to obtain diffusion time, which were then changed to diffusion coefficients based on a reference of rhodamine 6G. Finally, the diameter of the samples was calculated from the Stokes-Einstein equation, assuming spherical shape of the samples.

2.3. Syntheses of Polymer Samples 2.3.1. Synthesis of PEG-PAsp and P(Asp-AP) PEG-PAsp was obtained by alkali hydrolysis of PEG-PBLA (Mw/Mn = 1.04) prepared by ring opening polymerization of BLA-NCA from mPEG-NH2 according to the previous report6, and its unimodal distribution was confirmed by aqueous SEC (Eluent: 10 mM phosphate buffer (PB) containing 500 mM NaCl (pH = 7.4). Flow rate: 0.75 mL/min). The degree of polymerization (DP) of P(Asp) segment in the block copolymer was determined from the peak intensity ratio of the protons of the poly(aspartic acid) side chain to the methylene protons in the PEG segment in the 1H-NMR spectrum measured in D2O at 25 ºC6. Here, PEG-PAsp sample with DP of PAsp segment of 77 was used throughout the experiments. Cy3- and Cy5-labeled PEG-PAsp samples were prepared by conjugating Cy3 and Cy5, respectively, at the ω-chain end of the PAsp segment according to the previous procedure6. P(Asp-AP) was obtained by aminolysis reaction of PBLA with 1,5-diaminopentane (DAP)6. PBLA (Mw/Mn = 1.05) was prepared by ring opening polymerization of BLA-NCA using n-butylamine as an initiator. DP of P(Asp-AP) was determined from the peak intensity ratio of the methylene protons of butyl amine to the protons


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of side chain of P(Asp-AP) in the 1H-NMR spectrum measured in D2O at 25 ºC6. P(Asp-AP) sample with DP of 76 was used throughout the experiments, and its unimodal distribution was confirmed by aqueous SEC (Eluent: 10 mM acetic acid containing 500 mM NaCl. Flow rate: 0.75 mL/min) Details of synthetic procedures are described in the Supporting Information S1 and S2.

2.3.2. Synthesis of guanidinylated P(Asp-AP) (P(Asp-AP1-x/GPx)) P(Asp-AP1-x/GPx) (x: ratio of guanidinium groups in the side chain) was synthesized as follows: The primary amino groups in the side chain of P(Asp-AP) prepared as described in Section 2.3.1. were converted to guanidinium groups by the reaction with DMGP. Briefly, P(Asp-AP) (15 mg) and DMGP (696 mg, 50 eq. to primary amino groups in the side chain of P(Asp-AP)) were dissolved separately in 0.5 M Na2CO3 aq., of which pH was adjusted to 9.5 using 1 M HCl aq. Then, the P(Asp-AP) solution was added with the DMGP solution, and stirred for t h at 40 °C (t = 0.25–0.90). The reaction mixture was dialyzed against 0.01 M HCl for one day at room temperature, and then at 4 °C for 2 days, and finally against de-ionized water for 1 h. The resulting solution was lyophilized to collect the final product (13–16 mg, average yield 89%). Guanidinylated degree, x (ratio of guanidinium groups to amino groups in the side chain of P(Asp-AP)), was determined from the peak shift of methylene protons adjacent to primary amino group from 2.90-3.00 ppm to 3.05-3.25 ppm upon the conversion to guanidinium group in the 1

H-NMR spectra measured in D2O at 25 ºC (Supplementary Figure S1). The DP of P(Asp-AP1-

x/GPx)

was determined to be 76 from the peak intensity ratio of the methylene protons of butyl

amine to the protons of side chain of P(Asp-AP1-x/GPx) in the 1H-NMR spectrum measured in


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D2O at 25 ºC, and its unimodal distribution was confirmed by aqueous SEC (Eluent: Deionized water containing 3 M guanidine hydrochloride. Flow rate: 0.5 mL/min). A series of gunidinylated P(Asp-AP) with varying x was abbreviated as P(Asp-AP1-x/GPx).

2.4 Preparation and Characterization of PIC Assemblies PEG-PAsp and P(Asp-AP1-x/GPx) (0.15 ≤ x ≤ 0.90) were separately dissolved in 10 mM phosphate buffered saline (pH = 7.4; [NaCl] = 150 mM) to prepare the 1mg/mL solution. The P(Asp-AP1-x/GPx) solution was then added to the PEG-PAsp solution, and the mixture was stirred by a vortex mixer for 2 min at room temperature. The mixing charge ratio (y) of the polymers was defined by the following equation: y = [the total residual molar concentration of amino groups and guanidinium groups in the solution] / [the residual molar concentration of carboxyl groups in the solution]. Total concentration of the polymer (PEG-PAsp + P(Asp-AP1x/GPx))

in the mixed solution was set to the constant value of 1 mg/mL for all the experiments

done at varying y. For the preparation of non-guanidinylated PICsome from PEG-PAsp and P(Asp-AP), these polymers were separately dissolved in 10 mM phosphate buffer without NaCl to prepare 1 mg/mL solution, followed by the vortex mixing at y = 1 for 2 min at room temperature. Cy3- or Cy5-labeled PICsomes were prepared by using Cy3- or Cy5-labeled PEGPAsp (See Section 2.3.1. for the preparation) instead of PEG-PAsp. Detailed physicochemical characterization of PIC assemblies thus prepared was conducted by the use of DLS, TEM, and FCS as described in Sections 2.2.3., 2.2.4, and 2.2.5, respectively.


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2.5. Calculation of the residual molar ratio of cationic groups to anionic groups in PICsome. PICsome solution (Total concentration of the polymer: 1 mg/mL) was transferred into a thick wall ultracentrifuge tube (Beckman Coulter, Inc., USA) and set in an ultracentrifuge, Optima MAX-TL (Beckman Coulter Inc., USA), equipped with TLA120.1 rotor. After the rotation at 50000 g for 1.5 h, PICsome fraction was sedimented in the bottom of the tube. Then, the supernatant was collected to quantify the amount of free polymer fraction in the solution by measuring the UV absorption at the wavelength corresponding to amide bond (215 nm), allowing to determine the actual residual molar ratio of cationic groups to anionic groups in the PICsome (y’).

2.6. Evaluation of Blood Circulation Profile of PICsomes Balb/c mice (n = 3 for each sample) were anesthetized and set under the intravital real-time confocal laser scanning microscopy (IVRT-CLSM; Nikon AR1) for the observation of earlobe capillaries16. Then, 200 µL of Cy5-labeled PICsome solution (Total concentration of the polymer: 1.0 mg/mL) was systemically injected via the tail vein. 10 mM phosphate buffer with or without 150 mM NaCl was used for the preparation of injecting solution of guanidinylated or non-guanidinylated PICsomes, respectively. The change in the fluorescent intensity of Cy5 in the vein of capillaries was measured continuously over time until 20 min. The intensity was expressed in the relative value by normalizing against the maximum fluorescent intensity observed in the blood vessels just after the injection. Estimated concentration of PICsome in the blood pool of mice was calculated based on the whole blood volume (1.8 mL) described in the


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literature17. All animal procedures in this study were approved by the Animal Care and Use Committee of the Innovation Center of NanoMedicine, Kawasaki Institute of Industrial Promotion (Kanagawa, Japan).

3. Results and Discussion 3.1. Synthesis of a series of guanidinylated P(Asp-AP) with varying substitution degree Primary amino groups in the side chain of P(Asp-AP) (DP = 76) were converted to guanidinium groups by reacting with 3,5-dimethyl-1-guanylpyrazole nitrate (DMGP) in aqueous medium (Fig. 1a). The conversion ratio of amino group to guanidinium group was calculated from the peak shift in the1H-NMR spectrum of methylene protons adjacent to primary amino group in the side chain from 2.90-3.00 ppm to 3.05-3.25 ppm upon the conversion to guanidinium group (Supplementary Fig. S1). As shown in Fig. 1b, the conversion reaction follows first-order kinetics because of the large excess of DMGP, and eventually, the samples with varying guanidinylation rate can be prepared just by changing the reaction time (Supplementary Table S1). A series of guanidinylated polymers thus obtained were denoted as P(Asp-AP1-x/GPx) (0.15 ≤ x ≤ 0.90), where x stands for the ratio of the guanidinylated side chain in the polymer, and was used for PICsome preparation as described in the following Section.


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Figure 1. (a) Synthetic scheme of guanidinylated P(Asp-AP) (P(Asp-AP1-x/GPx)). (b) Change in the percentage of remaining amino groups in the side chain of P(Asp-AP) over the reaction time determined by 1H-NMR.

3.2. Characterization of PIC structure prepared under physiological salt condition Polyion complexes (PICs) were prepared under room temperature by mixing PEG-PAsp (Mw of PEG = 2,200；DP of PAsp = 77) and a series of P(Asp-AP1-x/GPx) (0.15 ≤ x ≤ 0.90) with varying guanidinylation ratio at a charge stoichiometric condition in 10 mM phosphate buffered saline (pH7.4; [NaCl] = 150 mM). Total concentration of the polymer (PEG-PAsp + P(Asp-AP1x/GPx))

in the mixed solution was set to the constant value of 1 mg/mL for all the combinations.

As we reported previously6, stable and mono-dispersed vesicular structure with unilamellar PIC membrane (PICsomes) was obtained from the combination of PEG-PAsp and P(Asp-AP) in 10 mM phosphate buffer without NaCl, yet the addition of 150 mM NaCl induced the transition to


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micrometer-scaled and polydispersed PIC assemblies10, 18, 19. Of interest, guanidinylation of P(Asp-Ap) exerted a substantial effect on this assembly scheme of PEG-PAsp/P(Asp-AP) to maintain the unilamellar PICsome structure even in phosphate buffered saline with 150 mM NaCl, as confirmed by TEM imaging (Fig. 2), indicating that the presence of guanidium groups in the side chain of polycation contributes to stabilize the lamellar structure of PIC, presumably due to hydrogen bond formation. Even a partial substitution of primary amino groups to guanidium groups in the side chains of P(Asp-AP), as low as ~15%, was enough to stabilize the vesicular structure in phosphate buffered saline. Furthermore, when half of the primary amino groups were replaced by guanidinium groups (x = 0.50), the size of PICsomes was reduced to approximately 100 nm with narrow distribution (Nano-PICsomes) (Fig. 2c) (See also DLS measurement results in the legend to Figure 2). On the other hand, when x < 0.50, the PICsomes were nearly in the micrometer range with broad distribution (Fig. 2a, b). Nano-PICsomes were also observed in the region of x > 0.50, yet some larger-sized PICsomes with diameter over 100 nm were always observed as a co-existing fraction (Fig. 2d, e). Note that, according to our previous papers7, 11, all the PIC samples were crosslinked before TEM observation to avoid their morphological changes which might be induced by the removal of phosphate and NaCl in the buffer and/or the process of staining and observation. Hereafter, to examine the future utility of guanidinylated PICsomes as systemically injectable drug carriers and nanoreactors, we placed the focus on the region that gives Nano-PICsomes (x ≥ 0.50) because of the significance of this size for achieving longevity in blood, while circumventing the non-specific retention in reticuloendothelial system organs such as spleen20.


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Figure 2. Representative TEM images of PICsomes composed of PEG-PAsp and P(Asp-AP1x/GPx)

with varying x at charge stoichiometric condition. All samples were crosslinked before

TEM observation to avoid morphological changes induced by the process of purification, staining, and observation. The size and the distribution of each sample determined by DLS are shown in the parenthesis as follows: (a) x = 0.15 (average diameter = 489 nm, PDI = 0.297), (b) x = 0.35 (average diameter = 254 nm, PDI = 0.323), (c) x = 0.50 (average diameter = 120 nm, PDI = 0.076), (d) x = 0.70 (average diameter = 138 nm, PDI = 0.225), and (e) x = 0.80 (average diameter = 118 nm, PDI = 0.182). Then, we deviated the mixing ratio of PEG-PAsp with P(Asp-AP1-x/GPx) (0.50 ≤ x ≤ 0.90) from the point of stoichiometric charge to increase the ratio of PEG-PAsp since the guanidium group has two binding sites for oxyanions, such as carboxylate groups, by forming multiple hydrogen bonding21-23, possibly leading to the formation of non-charge stoichiometric PIC structures. Here,


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the residual molar ratio of cationic groups (primary amino groups and guanidium groups in P(Asp-AP1-x/GPx)) to anionic groups (carboxylic groups in PEG-PAsp) in the mixture was defined as y (y = [the total residual molar concentration of amino groups and guanidinium groups] / [the residual molar concentration of carboxyl groups]), and the PIC samples with various combinations of x and y were prepared from PEG-PAsp/P(Asp-AP1-x/GPx)) for the characterization. Note that the lower limit of y was set at 0.6 to keep the weight fraction of PEG in the polymer mixture (fPEG) to be less than 10% to circumvent steric hindrance between PEG strands, particularly on the inner side of the PICsome, satisfying the condition for the formation of stable vesicle with PIC lamellar structure as determined previously24. The size distribution and the morphology of the obtained PICsomes from the mixture of PEGPAsp and P(Asp-AP1-x/GPx) (0.50 ≤ x ≤ 0.90) at varying y (0.60 ≤ y ≤ 1.00) were examined by DLS and TEM, respectively. Eventually, the TEM images clearly confirmed the formation of Nano-PICsomes in all of the observed points, yet the assemblage seems to be divided into two regions, depending on the dispersity of the PICsomes (Fig. 3a), and the region defined by the closed circles consists only with monodispersed Nano-PICsomes (Fig. 2c, 3b). Size of these Nano-PICsomes determined by DLS seems to be chiefly related with x as summarized in Supplementary Figure S2 (Supporting Information), and decreased from 120 nm at x = 0.50 to the range of 80~90 nm at x ≥ 0.70. Monodispersity of all of these Nano-PICsomes in the region of closed circles was confirmed also from DLS measurements (PDI < 0.1) (Data not shown). Hereafter, these monodispersed Nano-PICsomes were denoted as GSx-PICsomes with x being the ratio of guanidinium group in the side chain of polycation, P(Asp-AP1-x/GPx).


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Figure 3. (a) Diagram of PICsomes classified into two groups depending on their size distribution; the closed circled points represent the region with only mono-dispersed vesicles (GSx-PICsomes) and the open squared points represent the co-existing region of Nano-PICsomes and larger-sized PICsomes. (b) Representative TEM image of GSx-PICsomes (x = 0.70, y = 0.80). To get further insight into the composition of PICsomes obtained in the region depicted in Fig. 3a, the actual charge ratio (y’) in the PICsomes was estimated by quantifying the amount of the remaining polymers in the supernatant after the sedimentation of the PICsome fraction by ultracentrifugation. Interestingly, y’ kept as unity for all the PICsomes regardless of the mixing charge ratio (y), indicating the maintenance of charge stoichiometry in the structure of PICsomes with excess PEG-PAsp remaining in the free form in the solution (Supporting Information Table S2). This is a somewhat inconsistent observation to the assumption that guanidium groups may accommodate carboxylate groups over stoichiometry due to the capacity to form multiple hydrogen-bonding. Presumably, pairing with polyanion and polycation strands may be strong enough to keep charge-stoichiometric lamellar structure with proper alignment in PIC


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membrane, repelling the excess insertion of PEG-PAsp into the membrane to form hydrogen bonding network among guanidium and carboxylate groups. The presence of a region giving the mixture of Nano-PICsomes and larger-sized PICsomes with PDI value > 0.1 by DLS measurements (Data not shown) is somewhat puzzling (Fig. 3a; open squares), considering that, as summarized in Supplementary Table S2, actual charge ratio (y’) in PICsomes are still kept as unity, meaning the charge stoichiometry composition, even in this region. Interestingly, the boundary between the region where only Nano-PICsomes forms (Fig. 3a, closed circles) and the area where Nano-PICsomes plus larger-sized PICsomes are present (Fig. 3a; open squares) is a diagonal line, connecting y = 1.0 and x = 0.90. The mechanism involved in this boundary formation is yet to be clarified, but apparently, the region with open squares extended to lower y, meaning more deviation from the charge stoichiometry of the mixture (direction of excess PEG-PAsp), with an increased fraction of guanidinium groups in the polycation, suggesting a progressive increment of guanidinium groups in the polycation strand may be a key to induce this apparent change in the diagram of PICsomes. Of interest, as shown in Fig. 4a, b, and c, the further addition of P(Asp-AP0.30/GP0.70) to the GS0.70-PICsome solution at y = 0.8, settling in the region of closed circle, for shifting y to unity, corresponding to the region of open square, led to a uniform increase in the size of the GS0.70PICsome with maintaining the unimodal distribution from 86 nm (PDI = 0.065) to 152 nm (PDI = 0.089) according to DLS measurement. Appealing from the practical viewpoint is that one can manage the size of GSx-PICsomes initially formed in the region of closed circles just by controlling the additional amount of P(Asp-AP1-x/GPx) without impairing their monodispersity. Most plausible mechanism involved in this uniform increase in the size of GSx-PICsomes by P(Asp-AP1-x/GPx) addition might be an insertion of a single polycation/polyanion pair (unit PIC),


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Biomacromolecules

formed through the interaction of PEG-PAsp in the solution with newly added P(Asp-AP1-x/GPx), into the PIC membrane of the pre-existing GSx-PICsomes to increase the surface area, and eventually, the size as schematically shown in Fig. 4d. This mechanism of vesicle growth was reported in detail in our previous publication as a scheme of supramolecular polymerization7, and we assume the similar mechanism may play a role in the present system of PEG-PAsp/P(AspAP1-x/GPx). Notable feature here is an apparent discrepancy in the size and the distribution of PICsomes prepared through the different route of polyanion/polycation mixing even though they have the same final mixing ratio: The unimodal PICsome formation with average size of 152 nm (Fig. 4c) via the route depicted in Fig. 4a vs the polymodal PICsome formation with the mixture of Nano-PICsomes and micrometer-scaled vesicles (Fig. 2d) via the direct mixing of PEG-PAsp and P(Asp-AP0.30/GP0.70) at y = 1.0. To examine further this apparent discrepancy in the final PICsome structure depending on the preparation route, we then deviated y in an opposite direction to the one shown in Fig. 4a, i.e. adding PEG-PAsp to PEG-PAsp/P(Asp-AP0.30/GP0.70) PICsome solution at y = 1.0 so as to shift y into the region of the closed circles (y = 0.8) (Fig. S3 in the Supporting Information). Notably, in this case, there was no change in the size and the distribution of PICsomes, and they kept their considerably polydispesive nature, even settling in the region of the closed circles (before PEG-PAsp addition: average size =138 nm, PDI = 0.225; after PEG-PAsp addition: average size = 132 nm, PDI = 0.198). Further worthy to mention is that the average size and the distribution were decreased to 87 nm (PDI = 0.076) after 2 min of vortex mixing, corresponding to the formation of GS0.70-PICsomes. This apparently curious discrepancy in the characteristics of PICsomes depending on the preparation route may suggest an involvement of kinetically controlled regime in the formation of PICsome from the mixture of PEG-PAsp and (Asp-AP1-x/GPx). Although further insight in this unique kinetics of PICsome


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formation is worth to study from a physicochemical viewpoint, we hereafter focus to the main purpose of this study to evaluate the feasibility of GSx-PICsomes, Nano-PICsomes formed in the closed circle region, as vesicular carriers used in harsh in vivo conditions. Particularly, GS0.70and GS0.80-PICsomes were subjected to further examination because of their size (

Robust Polyion Complex Vesicles (PICsomes) under Physiological

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