Self-Assembly of pH-Responsive Microspheres for Intestinal Delivery

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Self-Assembly of pH-Responsive Microspheres for Intestinal Delivery of Diverse Lipophilic Therapeutics Xing Zhou, Yang Zhao, Siyu Chen, Songling Han, Xiaoqiu Xu, Jia Wei Guo, Mengyu Liu, Ling Che, Xiaohui Li, and Jianxiang Zhang Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b00512 • Publication Date (Web): 11 Jul 2016 Downloaded from http://pubs.acs.org on July 12, 2016

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Self-Assembly

Biomacromolecules

of

pH-Responsive

Microspheres

for

Intestinal Delivery of Diverse Lipophilic Therapeutics Xing Zhou,†, §,‡ Yang Zhao,†, ‡ Siyu Chen,† Songling Han,† Xiaoqiu Xu,† Jiawei Guo,† Mengyu Liu,† Ling Che,ǁ Xiaohui Li,*,§ and Jianxiang Zhang*,† †

Department of Pharmaceutics, College of Pharmacy, Third Military Medical University, Chongqing 400038,

China §

Institute of Materia Medica, College of Pharmacy, Third Military Medical University, Chongqing 400038,

China ǁ

Department of Pharmacy, Hospital 309 of PLA, Beijing 100091, China

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KEYWORDS: host-guest interaction • self-assembly • microsphere • pH-responsive • intestinal drug delivery • hydrophobic drug

ABSTRACT: Targeted delivery of therapeutics to the intestine is preferred for the management of many diseases due to its diverse advantages. Currently, there are still challenges in creating cost-effective and translational pH-responsive microspheres for intestinal delivery of various hydrophobic drugs. Herein we report a multiple noncovalent interactions-mediated assembly strategy, in which carboxyl-bearing compounds (CBCs) are guest molecules, while poly(N-isopropylacrylamide) (PNIPAm) serves as a host polymer. Formation of microparticles and therapeutic packaging can be achieved simultaneously by this assembly approach, leading to well-shaped microspheres with extremely higher drug loading capacity as compared to microspheres based on two FDA-approved materials of poly(D,L-lactide-co-glycolide) (PLGA) and a enteric coating polymer Eudragit®S 100 (S100). Also, carboxyl-deficient hydrophobic drugs can be effectively entrapped. These assembled microspheres, with excellent reconstitution capability as well as desirable scalability, could selectively release drug molecules under the intestinal conditions. By significantly enhancing drug dissolution/release in the intestine, these pH-responsive assemblies may notably improve the oral bioavailability of loaded therapeutics. Moreover, the assembled microspheres possessed superior therapeutic performance in rodent models of inflammation and tumor over the control microspheres derived from PLGA and S100. Therapy with newly developed microspheres did not cause undesirable side effects. Furthermore, in vivo evaluation in mice revealed the carrier material PNIPAm was safe for oral delivery at doses as high as 10 g/kg. Collectively, our findings demonstrated that this type of pH-responsive microspheres may function as superior and translational intestine-directed delivery systems for a diverse array of therapeutics.

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INTRODUCTION For the treatment of various diseases, oral drug delivery is preferred, due to its convenience, desirable cost-effectiveness, relatively good safety profiles, and high patient acceptance.1-3 Microspheres, derived from either natural or synthetic materials, have been widely studied for oral delivery of a wide variety of therapeutics.4-7 However, absorption of microparticulate delivery systems has been plagued by many physiological and physical barriers in the gastrointestinal (GI) tract, such as the intestinal mucus layer.8 This generally results in low and variable oral absorption efficiencies,9 and therefore rendering many encapsulated drugs ineffective after oral administration. To enhance drug absorption, physicochemical properties of carrier microspheres have been modulated, which include size,10 bulk properties (such as utilization of pH-responsive, biodegradable and/or mucoadhesive materials),11, 12 and surface chemistry (like surface hydrophobicity and surface charge).13 Besides, targeted delivery strategies have been investigated to further improve the absorption efficiency of microparticles, by physically absorbing or covalently conjugating antibodies,14, 15 ligands,16 or cell penetrating peptides,17 Also, biomimetic approaches have been employed to develop effective oral delivery carriers for various drugs.16, 18-20 Among these different strategies, considerable attention has been concentrated on pH-responsive delivery systems that can selectively release drug molecules at the intestinal sites.21-23 To realize selective drug release under

intestinal

conditions,

materials

that

can

undergo

degradation/hydrolysis,

swelling,

or

hydrophobic/hydrophilic transition at pH 6~8 are generally employed to construct oral delivery systems. The used materials include acrylic-based polymers (such as enteric coating polymer Eudragit®S 100, i.e. S100), alginate, and poly(γ-glutamic acid) as well as their copolymers,24-28 which may be processed into microparticles by self-assembly, emulsion methods, or other advanced technologies.10,

29-31

Therapeutics varying from

small-molecular drugs, peptides, proteins, to nucleic acids can be delivered using microparticles derived from these polymers. Whereas significant progress has been achieved by these delivery systems, with respect to increasing drug absorption, targeting diseased sites in the GI tract, or inducing desirable mucosal immune response, few of them have been translated to the clinic so far.32, 33 Currently it remains highly challenging to develop effective and translational microspheres for targeted drug delivery in the intestine. Firstly, it is difficult ACS Paragon Plus Environment

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to cost-effectively and massively synthesize polymer materials of interest with tailored molecular weight and/or structures, good quality control, and reproducible production,33 which are required for the development of qualified formulations with desirable benefit-risk ratios. Secondly, due to structural complexity and heterogeneity of currently developed pH-responsive materials, their response to pH fluctuation more often shows delayed and unsharp profiles, and therefore delicate control of drug release via pH is undesirable. Thirdly, there are challenges to facilely produce therapeutic microspheres with well-defined morphology, controlled size, excellent reconstitution capability, and desirable scalability. In addition, due to the relatively hydrophilic nature of most enteric coating materials with carboxyl, hydroxyl, or other polar moieties, their microparticles frequently display low drug loading capacity for many hydrophobic drugs, which is an additional issue limiting their practical applications.34 Consequently, there is still unmet demand for innovative strategies to create cost-effective and translational therapeutic microspheres for oral delivery of various drugs. To circumvent above issues and develop effective pH-responsive microspheres for intestinal drug delivery, herein we report a simple yet robust method that can afford therapeutic microspheres with well-defined structure, extremely high drug loading contents, notably enhanced drug dissolution, and dramatically improved bioavailability. This was achieved by a one-step route based on guest molecule-directed assembly of a structurally simple polymer via host-guest interactions. In this strategy, carboxyl-bearing compounds (CBCs) serve as guest molecules, while the host polymer is poly(N-isopropylacrylamide) (PNIPAm). When CBCs are pharmacologically active compounds, therapeutic packaging and microsphere formation can be simultaneously achieved. Besides, the pH-sensitivity of these microspheres is mainly dominated by CBC molecules, which is distinctly different from generally studied pH-responsive delivery systems since their sensitivity is determined by carrier materials. Due to the availability of a large number of CBCs, pH-sensitivity of microspheres based on our new strategy can be easily modulated for different applications. In addition to constructing pH-responsive therapeutic microspheres and exploring their intestinal delivery capability for multiple drugs, their therapeutic performance was examined in rodent models of inflammation and tumor.

EXPERIMENTAL SECTION ACS Paragon Plus Environment

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Materials. Azodiisobutyronitrile (AIBN), N-isopropylacrylamide (NIPAm), fluorescein O-acrylate, N-methylacrylamide,

N-ethylacrylamide,

N-propylacrylamide,

N-hydroxyethyl

acrylamide,

N,N-dimethylacrylamide, N-tert-butylacrylamide, indomethacin (IND), sulindac (SUL), carrageen, and complete Freund’s adjuvant (CFA) were obtained from Sigma-Aldrich (USA). Ursodeoxycholic acid (UDCA) was provided by Aladdin Industrial Corporation (Shanghai, China). Poly(D,L-lactic-co-glycolic acid) (PLGA, 50:50) with intrinsic viscosity of 0.50-0.65 was purchased from Polysciences, Inc (USA). Eudragit S 100 (S100) was kindly supplied by Evonik Industries (Germany). Poly(vinyl alcohol) (PVA) (88 mol% hydrolyzed, Mw = 25 kDa) was obtained from Acro Organics. Paclitaxel (PTX) was obtained from Xi’an Xuan Biological Technology Co., Ltd (Xi’an, China). All the other reagents are commercially available and used as received. Synthesis of Polymers. Poly(N-isopropylacrylamide) (PNIPAm) was produced by free radical polymerization according to the previously reported method.35 Briefly, 5.0 g NIPAm in 30 mL of anhydrous methanol was polymerized in the presence of 0.15 g AIBN at 60°C for 24 h. After precipitation from diethyl ether, PNIPAm was purified by dialysis, and then freeze-dried. The similar methods were followed to synthesize fluorescein-labeled PNIPAm by copolymerization of fluorescein O-acrylate and NIPAm. In this case, the molar ratio of NIPAm/AIBN and NIPAm/fluorescein O-acrylate was 50:1 and 100:1, respectively. Following the same protocols, other N-substituted polyacrylamides including poly(N-methylacrylamide), poly(N-ethylacrylamide), poly(N-propylacrylamide),

poly(N-hydroxyethylacrylamide),

poly(N,N-dimethylacrylamide),

and

poly(N-tert-butylacrylamide) were synthesized. To measure Mw of PNIPAm and fluorescein-labeled PNIPAm, gel permeation chromatography (GPC) was carried out using a Waters model 1515 system, equipped with a Waters 2414 refractive index detector. THF was used as a mobile phase, and Mw calibration was conducted with polystyrene standards. Preparation of Various Assembled Microspheres. Assembly of microspheres was performed by a dialysis procedure. In brief, different pairs of CBC/PNIPAm at defined feeding ratios were dissolved in dimethyl sulphoxide (DMSO), with the PNIPAm concentration of 10 mg/mL. Thus obtained solution was dialyzed against deionized water at 25°C. The outer aqueous solution was refreshed every 2 h. Post 24 h, samples were harvested for additional analysis without other treatments. The similar procedures were implemented for other ACS Paragon Plus Environment

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CBC/poly(N-alkyacrylamide) pairs, and also used to produce assemblies containing hydrophobic drugs without carboxyl. Preparation of Drug-Loaded Microparticles by an Emulsion Technique. An oil-in-water (o/w) emulsion solvent evaporation method was adopted to prepare PTX-containing microspheres derived from either PLGA or S100.31 For PLGA microspheres, both PLGA and drug were dissolved in dichloromethane (DCM) to obtain an oil phase. Aqueous solution of PVA (2 wt%) was used as a water phase. The o/w emulsion was achieved by homogenizing at 25, 000 rpm. After the solvent was evaporated at room temperature for 3 h, microspheres were harvested by centrifugation, and then washed with deionized water four times. In the case of S100 microspheres, the oil phase containing both S100 and drug was achieved by dissolving them in ethanol/DCM mixture (v:v = 1:3). Subsequently, the similar procedures were followed to produce PTX-loaded S100 microspheres. Quantification of Drug Loading Contents in Various Microparticles. To quantify drug contents in various microparticles, accurately weighed amount of freeze-dried microspheres was dissolved or dispersed in defined volume of methanol. Then the drug concentration in methanol was determined by UV measurements at 310 nm for IND and SUL, while the PTX content was quantified by high performance liquid chromatography (HPLC, LC-20A, Shimadzu). The drug loading content and entrapment efficiency was separately calculated according to the following equations.

Loading content (%) =

Weight of drug in microparticles × 100% Weight of microparticles

Entrapment efficiency (%) =

Drug content in microparticles × 100% Theoretical drug content

Characterization of Microparticles. Transmission electron microscopy (TEM) was conducted on a TECNAI-10 microscope (Philips, Netherlands), operating at 80 kV. Formvar-coated copper grids were used. Specimens were prepared by dipping the grid into aqueous solutions of various microparticles, and extra solution was blotted with filter paper. After water was evaporated at room temperature, samples were observed directly without any staining. Scanning electron microscopy (SEM) images were acquired on a field emission scanning electron microscope (XL30FEG, Phillips). Samples were prepared by coating aqueous solutions of various particles onto freshly cleaved mica, and water was evaporated at room temperature under normal ACS Paragon Plus Environment

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pressure. Particle size and ζ-potential measurements were carried out on a Malvern Zetasizer Nano ZS instrument at 25°C. Differential scanning calorimetry (DSC) was performed on a TA2000 calorimeter (TA Instruments, USA). DSC curves were collected from the first heating run at 10°C/min under a nitrogen flow of 50 mL/min. Powder x-ray diffraction (XRD) patterns were obtained using a RIGAKU diffractometer. Confocal laser scanning microscopy (CLSM) images were acquired on a fluorescence microscope (Leica, Heidelberg, Germany). Analysis by Fourier transform infrared (FT-IR) spectroscopy was performed on a FT-IR spectrometer (Perkin-Elmer S100, USA). 1H NMR spectrometry was conducted on a Varian INOVA-400 spectrometer at 400 MHz. Molecular Modeling of Microspheres Formation. The repeat structural units of PNIPAm were built in a three-dimensional (3D) coordinate using a Molecular Operating Environment software package builder tool (MOE’s, Chemical Computing Group, Canada).36 The polymer chain was built through a head-to-tail connection with 50 structural units due to the software limitation. PNIPAm with 5 structural units was employed as a short-chain polymer to simulate intermolecular interactions in the docking process. The 3D structures of IND, PNIPAm, PTX, and water molecules were preoptimized before running simulations using an all-atom MMFF94x force field with no constraints. Then, IND, PTX, PNIPAm segments with 5 repeating units, and water molecules preoptimized were docked into the minimized, hydrated polymer structure and small molecules of interest using an AutoDock software package (AutoDock4.2, the Scripps Research Institute) to estimate the binding energy and intermolecular energies.36, 37 The size of the grids was set at 126×126×126 Å using grid spaces at 0.375 for all docking calculations. Four search algorithms (including Genetic Algorithm, GA; Lamarckian Genetic Algorithm, LGA; Simulating Annealing, SA; and Local Search, LA) built in Autodock4.2 were probed to find the most favorable drug-polymer complex geometry. For all simulations, docking runs were set to 100. For GA and LGA searching algorithms, the number of energy evaluation was set to 2.5×107, while the population size was 150. The other docking parameters were set to the default values for all algorithms. Since the binding sites within PNIPAm are not defined, blind docking was applied to the entire polymer chain and small molecules of interest. The docking

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results were analyzed by Autodock tools (ADT 1.56). Since we found that LGA predicted the strongest affinity between PNIPAm and small molecules (Table S1), this algorithm was used in following simulations. To simulate changes in conformation and intermolecular interactions when IND, PTX, and PNIPAm were in one system, IND-docked PNIPAm with the lowest energy conformation was acquired, which served as the host for docking with PTX to achieve the lowest energy conformation of IND/PTX. Subsequently, PNIPAm and IND/PTX were employed to conduct molecular dynamics (MD) simulations in water environment, using a canonical ensemble with a target temperature of 300 K and an integration time step of 1 fs within a total simulation time of 5 ns. Similar procedures were followed to simulate interactions of PTX/PLGA and PTX/S100. In these cases, PLGA and S100 with 5 structural units were employed. In Vitro Release Study. About 10 mg of freeze-dried microspheres was reconstituted in 30 mL of 0.01 M PBS at pH 7.4 and incubated at 37°C. At different time points, 4.0 mL of PBS was collected, and the same volume of fresh PBS was supplemented. Also, in vitro drug release was examined in solutions simulating pH conditions in the GI tract. For this purpose, the release test was initially conducted at pH 1.2. After 2 h, the release medium was switched into PBS at pH 7.4. The drug concentration in the release buffer was quantified by UV in the case of IND and SUL, while HPLC was employed to quantify PTX concentrations. Animals. Male Sprague-Dawley (SD) rats (200-250 g), Kunming mice (20-25 g), and Balb/C athymic nude mice (18-22 g) were obtained from the Animal Center at the Third Military Medical University. Animals were housed in standard rat/mouse cages under conditions of optimum light (12 h light-12 h dark cycle) and temperature at 22 ± 1°C, with ad libitum access to water and food. All the animals were acclimatized to the laboratory for at least 3 days before experiments. All the animal care and experimental protocols were performed in compliance with the Animal Management Rules of the Ministry of Health of the People's Republic of China (No. 55, 2001) and the guidelines for the Care and Use of Laboratory Animals of Third Military Medical University. In Vivo Pharmacokinetic Study. To examine in vivo pharmacokinetic profiles of various IND microparticles, 25 male SD rats were randomly divided into five groups with 5 rats in each group. Oral administration of microparticles of IND/PLGA (at 5 mg/kg of IND) and IND/S100 (at 5 mg/kg of IND) as well ACS Paragon Plus Environment

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as IND/PNIPAm assemblies at 1.25, 2.5, and 5.0 mg/kg of IND were separately carried out via gastric gavage. All rats were fasted overnight before administration. After gavage, blood samples were collected at different time points. Similar procedures were followed to study pharmacokinetic behaviors of SUL (at 5 mg/kg) and PTX (at 10 mg/kg). Drug concentrations in the plasma were quantified by HPLC according to previously established methods.23, 38 The values of area under the plasma drug concentration-time curve (AUC) were calculated according to a non-compartmental model using the software of DAS 3.1.0. In Vivo Efficacy of IND/PNIPAm Microspheres in Inflammatory Disease Models. Therapeutic effects of assembled IND/PNIPAm microspheres were evaluated in both acute and chronic models of inflammation. The acute model was established by intradermal (i.d.) injection of 100 µL of carrageen solution (0.1 wt% in saline) in the right-hind paw of male SD rats. After 30 min, saline, IND/PLGA and IND/S100 microparticles (at 5 mg/kg of IND), and IND/PNIPAm microspheres (at 1.25, 2.5, and 5.0 mg/kg of IND) were orally administered (n = 6 in each group), respectively. After administration, the volume of right-hind paw was determined at various time points. The edema degree was expressed as difference in the paw volume before and after inflammation. The chronic model was induced by i.d. injection of CFA. After one week, different IND microparticles were administered by oral gavage every three days, and the dosing regimen was same as that employed in the acute model. Subsequently, similar procedures were implemented to evaluate therapeutic benefits of IND/PNIPAm microspheres. To examine possible side effects, the body weight of rats was monitored before and after experimentation. In addition, GI tissues and major organs (including heart, liver, spleen, lung, and kidney) were resected from rats subjected to various treatments, after they were euthanized. All tissues were fixed in 4 wt% paraformaldehyde. Histopathological sections were prepared and stained with hematoxylin and eosin (H&E). All the histological sections were imaged by optical microscopy. Antitumor Activity of Assembled PTX Microspheres. B16F10 murine melanoma cells were cultured in 96-well plates at a density of 1.0 × 104 cells/well in 100 µL of growth medium containing 10% (v/v) fetal bovine serum (FBS), 100 U/mL of penicillin, and 100 µg/mL of streptomycin. Cells were incubated at 37°C in a humidified atmosphere of 5% CO2. To generate B16F10 tumor xenografts, 1 × 107 cells suspended in 0.1 mL of ACS Paragon Plus Environment

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DMEM containing 10% FBS were inoculated into the right limb armpits of athymic mice (4-6 weeks old). After 10 days, when the average volume of tumor xenografts reached ~300 mm3, the mice were randomly assigned into 5 groups, with 6 mice in each group. Various formulations including saline, and microspheres of PTX/PLGA, PTX/S100, PTX/IND/PNIPAm, and PTX/SUL/PNIPAm were orally administered every four days. The dose of PTX was 10 mg/kg in all groups of PTX-containing microparticles. Fourteen days post treatment, the mice were euthanized. Blood samples were collected for further analysis. Main organs including heart, liver, spleen, lung, and kidney were excised and weighed. Also, GI tissues were harvested to examine possible irritations on the GI tract. For histopathological analysis, tissues were fixed in 4 wt% paraformaldehyde, and then sections were prepared and stained with H&E. Measurement of Clinical Chemistry Parameters. The blood samples were harvested into EDTA spray-coated tubes. The plasma concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (UREA), and creatinine (CREA) were determined (Roche Cobas C501, Roche Co., Switzerland). Toxicological Study of PNIPAm for Oral Delivery. The preliminary acute toxicity test was implemented for PNIPAm in mice. Specifically, PNIPAm in saline was orally administered at 0, 1.25, 2.5, 5.0, 7.5, 10.0 g/kg (5 mice in each group), respectively. After administration, the body weight and behaviors of mice were monitored. At day 14, all mice were euthanized. Major organs including heart, liver, spleen, lung, and kidney were resected for histological sections, followed by staining with H&E. Blood samples were collected for analyses of hematological parameters and biochemical markers related to liver/kidney functions. Statistical Analysis. Statistical analysis was performed using an one-way ANOVA test for experiments containing more than two groups, and with a two-tailed, unpaired t-test in experiments with two groups. The value of p < 0.05 was considered to be statistical significance.

RESULTS AND DISCUSSION Host-Guest Recognition Mediated Assembly of CBC/PNIPAm Microspheres. As well known, water-insoluble drugs are generally packaged into delivery systems such as liposomes, polymeric micelles, ACS Paragon Plus Environment

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nanoparticles, and microparticles by hydrophobic interaction between therapeutics and carrier materials,32, 39-41 with the exception of covalent conjugation.42 In most cases, however, this relatively weak interaction cannot overcome non-covalent forces among drug molecules that may mediate their aggregation/crystallization,43, 44 resulting in the low drug proportion incorporated into hydrophobic domains of different carriers, especially when high drug feedings are concerned.45, 46 This is mainly responsible for the low drug loading capacity of most particulate delivery systems. Under certain conditions, the low drug content might increase the risk of potential toxicity associated with the overdose of carrier materials. Consequently, there is still unmet demand for innovative loading strategies to effectively entrap lipophilic therapeutics into microparticles. Most recently, we found that CBCs-directed assembly of NIPAm homopolymer and its copolymers is a facile but effective route to create nanoparticles and microspheres with controlled superstructures over length scales.47 Through this strategy, a wide variety of pharmacologically active CBCs can mediate PNIPAm assembly, giving rise to well-defined microspheres (Scheme 1). Accordingly, this host-guest assembly approach represents a promising route towards highly effective polymeric microspheres for a diverse array of therapeutics. Herein we speculate that pH-responsive microparticles can be developed by CBC-mediated PNIPAm assembly, in consideration of pH-dependent dissociation and solubility switching of CBCs in aqueous solution. In addition, this strategy has the potential of offering microparticles with high drug loading capacity, since particle formation and therapeutic loading are simultaneously accomplished during assembly. Initially IND was used as a CBC, which is a nonsteroidal anti-inflammatory drug (NSAID) clinically used for the treatment of osteoarthritis, rheumatoid arthritis, and ankylosing spondylitis. IND employed in this study is mainly composed of the γ-form, exhibiting tablet-like crystals (Figure 1A). Dialysis of IND alone in DMSO against deionized water still produced aggregates of needle-like crystals (Figure 1B-C), with a melting point at 146.5°C (Figure S1A). The powder XRD pattern indicated that thus derived IND crystals belong to the α-form (Figure S1B). By contrast, when PNIPAm (Mw = 10 kDa) was used as a guest material, assembly of IND/PNIPAm at various weight ratios generated well-shaped microspheres (Figure 1D-I). The average size of IND/PNIPAm microspheres was gradually increased from 158.7, 486.5, 573.0, 627.8, 745.8, to 1042.0 nm, when their feeding ratio was enhanced from 0.2:1, 0.6:1, 0.8:1, 1:1, 2:1, and 3:1, respectively (Figure 2A). TEM ACS Paragon Plus Environment

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examination suggested that microspheres assembled at the high IND/PNIPAm ratios were relatively compact (Figure 1J). This was further affirmed by observation via CLSM, taking advantage of intrinsic blue fluorescence of IND (Figure 1K). Also, solid green microspheres were observed when IND was assembled with fluorescein-labeled PNIPAm (Figure 1L). Similarly, microspheres were assembled by PNIPAm and SUL (another NSAID generally used to treat acute or chronic inflammatory conditions as well as studied for tumor therapy)48 or UDCA (a FDA-approved drug utilized to treat gallstones and primary biliary cirrhosis),49 as illustrated in Figure 1M-P. Different from IND/PNIPAm microspheres that showed increased size with increase in the IND content, particle size of UDCA/PNIPAm assembled microparticles was decreased as the UDCA/PNIPAm weight ratio increased from 2:1 to 3:1. This difference might be attributed to different intermolecular interactions between IND-PNIPAm and UDCA-PNIPAm. According to molecular modeling, the intermolecular energy of UDCA-PNIPAm was -7.9 kcal/mol, which was much higher than that of IND-PNIPAm with -6.50 kcal/mol. Consequently, dense microspheres with smaller size would be assembled by UDCA/PNIPAm, when the content of UDCA was increased. By contrast, IND/PNIPAm assembled microspheres displayed a less compact structure as compared to those formed by UDCA/PNIPAm. As a result, particle size of IND/PNIPAm was increased with increase in the IND content. Nevertheless, further studies are necessary to fully elucidate this phenomenon. According to characterization by FT-IR and 1H NMR spectroscopy, this CBC-induced assembly of PNIPAm is driven by multiple interactions including hydrogen-bonding (H-bonding), hydrophobic, and electrostatic forces (Figure S2-3). H-binding mainly contributes to forces between carboxyl in CBC and amide of PNIPAm, while hydrophobic interaction exists among lipophilic moieties of CBC and PNIPAm. Besides, there are electrostatic interactions between PNIPAm and IND, due to the Lewis acid-base reaction. The synergetic effects of these noncovalent forces may conquer interactions among CBC molecules, thereby preventing their own aggregation or crystallization. Additionally, this host-guest assembly is strongly dependent on the structure of host polymers. For polyacrylamide derivatives with either more hydrophilic or more hydrophobic N-substituted groups than isopropyl, desirable coassembly could not be realized to afford microspheres upon dialysis with varied CBCs. For these polyacrylamide derivatives, considerable crystallization occurred during dialysis even at ACS Paragon Plus Environment

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the IND/polymer weight ratio of 2:1. As illustrated by representative SEM images (Figure S4), only IND crystals were formed for highly hydrophilic polymers such as poly(N,N-dimethylacrylamide) and poly(N-hydroxyethylacrylamide). On the other hand, both spherical microparticles and IND crystals could be observed in dialyzed samples of IND and poly(N-propylacrylamide) or poly(N-tert-butylacrylamide). Since coassembly of IND and N-substituted polyacrylamide is mainly dominated by a collective effect of H-bonding and hydrophobic interactions between guest IND molecules and polymer chains, these results suggested that the IND/PNIPAm system has optimal binding forces for assembly. Previously, theoretical calculation indicated that IND/PNIPAm has the largest intermolecular binding energy, among different IND/polymer pairs.47 Also, measurement

by isothermal

titration

calorimetry revealed

that

assembly of

IND/PNIPAm

was

thermodynamically favorable. Effective Loading of Various Therapeutics into Host-Guest Assembled Microspheres. Since microparticle formation and therapeutic packaging can be simultaneously achieved for pharmacologically active CBCs, this host-guest assembly rendered highly effective drug loading. For assembled IND/PNIPAm microspheres, the drug loading content was linearly increased as the theoretical IND feeding was enhanced, and the highest IND content was about 84% (Figure 2B). Moreover, the loading efficiency was higher than 90% independent of IND/PNIPAm ratios. According to the literature, this represents the highest drug loading content achieved so far for this hydrophobic drug. By contrast, assembly of IND with other poly(N-alkylacrylamide)s only gave rise to low drug loading. For instance, IND crystals could be observed when IND was assembled with poly(N-tert-butylacrylamide) even at a weight ratio as low as 1:5. Accordingly, similar to coassembly profiles, effective loading of guest CBC molecules is also closely related to the structure of host polymers. Subsequently, comparative experiments were conducted using carrier materials of PLGA (a FDA-approved hydrophobic and biodegradable polymer) and S100 (a FDA approved pH-sensitive acrylic-based polymer practically used for enteric coating formulations), both of them have been widely utilized to develop formulations for oral drug delivery.3, 50-53 IND-containing PLGA or S100 microspheres were prepared by an emulsion method. It was found that the highest IND loading content was about 15.7% and 14.6% (corresponding to the theoretical value of 20%) for PLGA and S100-derived microparticles, respectively. ACS Paragon Plus Environment

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Further increase in theoretical drug feeding during preparation of IND-loaded PLGA or S100 microparticles led to drug crystallization (Figure S5). Also, effective entrapment of SUL was realized by this assembly approach (Figure S6), and as high as ~80% of drug contents could be attained in resulting SUL/PNIPAm microspheres. To the best of our knowledge, this host-guest assembly denotes the most effective strategy that may give extremely high drug loading for this type of drugs. Besides desirable drug loading capacity, the procedures employed for fabricating these therapeutic assemblies displayed good scalability and reproducibility. For IND/PNIPAm and SUL/PNIPAm microspheres, tens to hundreds of grams of samples could be easily obtained in the laboratory. More importantly, as exemplified by IND/PNIPAm, thus assembled microspheres owned more excellent redispersibility in aqueous solution after freeze-drying. There was no significant aggregation or change in average size after reconstitution of lyophilized IND/PNIPAm microspheres (Figure S7). In contrast, emulsion-based PLGA or S100 microspheres were seriously aggregated, and it was difficult to achieve desirable reconstitution. These attributes are advantageous for bench-to-bedside translation of these therapeutic assemblies. Measurements by XRD and DSC suggested the absence of drug crystals in resulting assemblies (Figure 2C-D), demonstrating that drug molecules were amorphous in IND/PNIPAm microspheres. This is favorable for drug dissolution and thereby increasing the bioavailability. In Vitro Release and In Vivo Pharmacokinetic Studies. In vitro tests were then performed to examine release profiles of various drug-loaded microparticles. Under in vitro conditions simulating the GI tract, IND/PNIPAm microspheres of various feeding ratios showed almost no drug release at pH 1.2 (Figure 3A). Comparatively, considerably rapid release occurred at pH 7.4, and IND was almost completely released within 24 h. In addition, this release behavior was independent of drug loading, although the release rate was slightly slowed with increase in the IND loading content. The pH-responsive release profiles should be closely related to the characteristics of IND. At low pH, carboxyl of IND is protonated, leading to extraordinarily low solubility.54 In addition, at release temperature of 37°C that is higher than the lower critical solution temperature (LCST) of PNIPAm in water, PNIPAm chains become more hydrophobic due to dehydration of amide groups.29 The combined effect of hydrophobic IND and PNIPAm caused negligible IND release in the stomach-relevant acidic ACS Paragon Plus Environment

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solution. At pH 7.4, however, dissociation of IND can dramatically enhance its dissolution due to notably increased solubility at pH higher than its pKa of ~4.5.55 Moreover, the dissolution of IND may lead to erosion or disintegration of assembled IND/PNIPAm microspheres, further accelerating drug release. Consequently, these host-guest assemblies could selectively release cargo molecules under intestinal pH conditions, and therefore are promising for oral drug delivery. Additional release experiments were conducted using PLGA and S100 derived and IND-laden microspheres with comparable size as controls (Figure S8). Compared with IND/PLGA or IND/S100 microspheres produced by the emulsion technique, assembled IND/PNIPAm microparticles displayed significantly enhanced drug dissolution at pH 7.4 (Figure 3B). These findings also indicated that IND/PNIPAm assemblies exhibited more excellent pH-triggered release behaviors at pH 7.4 as compared to microspheres based on the enteric coating material S100. Subsequently, in vivo pharmacokinetic study was carried out in SD rats. Consistent with in vitro release results, oral administration of IND/PNIPAm microspheres at 5.0 mg/kg of IND (the high dose) caused markedly higher plasma drug concentrations in comparison to microspheres of IND/PLGA and IND/S100 at the same dose (Figure 3C). This resulted in remarkably enhanced AUC (Figure 3D). At 5.0 mg/kg of IND, the AUC value of IND/PNIPAm microspheres was 2.3 and 2.0 times of that corresponding to IND/PLGA and IND/S100 microparticles, respectively. The medium dose of IND/PNIPAm microspheres (2.5 mg/kg of IND) yielded an AUC that was 1.73 and 1.71 folds of that for IND/PLGA and IND/S100 microspheres at the high dose of 5 mg/kg of IND, respectively. Notably, even the low dose of IND/PNIPAm microspheres (1.25 mg/kg of IND) displayed plasma drug concentrations comparable to the high dose of IND/PLGA and IND/S100. Similarly, AUC of orally administered SUL was considerably enhanced by formulating into SUL/PNIPAm microspheres via assembly, when compared with PLGA or S100-based microspheres produced by the emulsion method. In this case, oral gavage of SUL/PNIPAm microspheres at 5.0 mg/kg of SUL resulted in the AUC value that was 6.1 and 2.2 times of that for SUL/PLGA and SUL/S100 microspheres, respectively (Figure S9). Consequently, above finding strongly suggested that CBC-mediated assembly of PNIPAm is a facile but robust approach to construct particulate delivery systems with high drug loading capacity, improved release performance, and enhanced bioavailability. Whereas PNIPAm and its copolymers have been widely utilized as ACS Paragon Plus Environment

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thermosensitive materials to fabricate delivery systems for different therapeutics varying from small molecules, peptides/proteins, to nucleic acids,29, 56, 57 herein we demonstrated for the first time that the hydrophilic PNIPAm itself can be employed as a useful host polymer to develop pH-triggerable microparticles, by taking advantage of strong interactions between PNIPAm and CBCs. Therapeutic Evaluations in Inflammatory Models in Rats. Above findings suggested that assembled CBC/PNIPAm microspheres may serve as promising oral delivery systems. To illuminate the therapeutic significance of these assemblies, we implemented in vivo pharmacodynamic studies in both acute and chronic inflammatory models. Whereas oral administration of various IND microparticles was able to mitigate the swelling degree of acute paw edema induced by carrageen in SD rats, the assembled IND/PNIPAm delivery system showed more potent activity (the left panel, Figure 4A). In particular, the low dose (1.25 mg/kg of IND) of IND/PNIPAm assemblies displayed desirable efficacy comparative to IND/PLGA and IND/S100 microspheres at the high dose of 5.0 mg/kg (see statistical analysis results in Table S2). Likewise, IND/PNIPAm microspheres achieved impressive therapeutic outcomes in the chronic arthritis model induced by CFA (the right panel, Figure 4A). In this case, IND/PNIPAm assemblies at the low (1.25 mg/kg) and medium (2.5 mg/kg) dose afforded efficacy comparable to that of IND/PLGA and IND/S100 microparticles at 5.0 mg/kg, respectively (see statistical analysis results in Table S3). In both cases, the high dose of IND/PNIPAm assemblies attenuated inflammation to a much greater extent than that of IND/PLGA or SUL/S100 microspheres at the same dose. The weight loss and GI irritation are major adverse effects associated with IND treatment.58 Whereas IND/PLGA and SUL/S100 groups exhibited significantly lower body weight gain as compared to the saline control, rats treated with IND/PNIPAm microspheres at various doses displayed normal increase in the body weight (the left panel, Figure 4B). In the chronic model, both PLGA and S100 groups possessed relatively low weight increase, and significant difference could be found between IND/PLGA and the saline control. Consistent with these findings, we did not observe abnormities in H&E stained pathological sections of stomach and proximal small intestine (both of them are susceptible to injuries post IND treatment) from IND/PNIPAm assemblies-treated animals at 5.0 mg/kg of IND (Figure 4C), in both acute and chronic models. Besides, no ACS Paragon Plus Environment

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evidently aberrant changes could be detected in H&E sections of major organs (including heart, liver, spleen, lung, and kidney) from rats subjected to various treatments (Figure 4D-E). These results strongly suggested that the host-guest assembly strategy not only remarkably improved pharmaceutical properties, but also notably potentiated therapeutic efficacy of encased drugs, when compared with particulate systems processed from FDA-approved hydrophobic or enteric materials by an emulsion process. Host-Guest Assemblies as Efficient Delivery Carriers of PTX. Above results clearly demonstrated that oral bioavailability of bioactive CBCs could be dramatically enhanced by assembly with PNIPAm. Considering the presence of a large number of carboxyl-deficient hydrophobic drugs with low oral bioavailability, we further examined whether these drugs can be loaded into this type of host-guest assemblies to improve their in vivo performance. Firstly, IND/PNIPAm assemblies were investigated as a proof of concept. PTX, a highly hydrophobic drug widely used for the treatment of diverse cancers and coronary artery disease,59, 60 was studied as a model carboxyl-deficient drug. Due to its low aqueous solubility, poor intestinal permeability, and efflux by P-glycoprotein, oral administration of particulate PTX formulations often results in low oral availability.61 Accordingly, innovative pharmaceutical strategies are desperately required to address this issue. Although dialysis of PTX solubilized in DMSO against deionized water generated crystals (Figure 5A and Figure S10), co-assembly of PTX with IND/PNIPAm produced well-defined microspheres when DMSO was used as a common solvent (Figure 5B-D). The size of PTX/IND/PNIPAm assemblies varied from 900 to 1300 nm as their weight ratio changed from 0.5:2:1 to 2:2:1 (Figure 5E). Likewise, PTX could coassemble with SUL/PNIPAm to form spherical assemblies (Figure S11). Quantification of the PTX content in PTX/IND/PNIPAm microspheres revealed its efficient loading, with the efficiency higher than 90% (Figure 5F). It is worth noting that the highest loading content of up to 60% could be achieved for this highly hydrophobic drug PTX. DSC measurements indicated that both the carboxyl drug and PTX were amorphous in these assemblies (Figure 5G). By contrast, the highest PTX loading content achieved for PLGA and S100 microspheres produced by the emulsion-based technique was about 9.8% and 1.3%, respectively; while significant crystallization occurred at relatively high drug feedings. Consequently, the host-guest assemblies based on CBC/PNIPAm may function as highly effective carriers for other hydrophobic therapeutics without ACS Paragon Plus Environment

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carboxyl. Similar to CBC/PNIPAm assemblies, the assembled PTX/IND/PNIPAm microspheres owned excellent reconstitution capability after freeze-drying. In addition, assembly of PTX/IND/PNIPAm microspheres could be easily scaled up for comprehensive preclinical evaluations, with good repeatability and reproducibility. All these features are additional advantages of this assembly approach to develop drug delivery systems that warrant their translation from the laboratory to the clinical trials. Molecular Simulation of Molecular Interactions in PTX-Containing Microspheres. Further, a computational approach was used to simulate assembly of PTX/IND/PNIPAm. We assumed that formation of PTX/IND/PNIPAm microspheres is mediated by intermolecular forces among three components which are stronger than interactions dominating drug aggregation/crystallization. Therefore, the Autodock program was used to estimate the values of binding energy and intermolecular energy between two components in the assembly system containing PTX, IND, PNIPAm, and water. The binding energy is usually used to measure the intermolecular affinity or interaction. Our docking studies revealed that IND, PNIPAm, and IND exhibited the strongest affinity to PTX, IND, and PNIPAm, respectively (Figure S12). This suggested that PNIPAm tends to interact with IND, while PTX prefers complexation with IND. Accordingly, IND may simultaneously interact with PNIPAm and PTX in this three components system of PTX/IND/PNIPAm. Then the lowest energy complexes of IND/PTX, PTX/PNIPAm, and IND/PNIPAm were rendered as 2D and 3D styles by MOE (Figure 6A-B), and their intermolecular energy was calculated by ADT. We found that forces between IND and PTX are mainly composed of π-π stacking and H-bonding between hydroxyl of PTX and carbonyl of IND, while intermolecular forces of PTX/PNIPAm are primarily resulted from hydrophobic interactions. On the other hand, H-bonding, electrostatic, and hydrophobic interactions largely contribute to IND-PNIPAm forces. Notably, the H-bonding energy of IND/PNIPAm is much higher than that of IND/PTX, leading to preferential interaction of PNIPAm/IND over PTX/IND via H-bonding. Accordingly, we assumed that IND may firstly interact with PNIPAm through H-bonding, and then residual moieties of IND may complex with PTX via H-bonding and π-π stacking. Besides, PTX will interact with exposed lipophilic units of PNIPAm. To verify this assumption, IND, PTX, and PNIPAm were successively docked as a PTX/IND/PNIPAm complex to perform MD simulation in ACS Paragon Plus Environment

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water environment. It was found that the PTX/IND/PNIPAm complex was stable in the simulated water environment. The three components showed a complicated H-bonding network to form a stable assembly (Figure 6C), in which hydroxyl and carbonyl of the carboxyl group in IND formed H-bonding with PTX and PNIPAm, respectively. This is consistent with our hypothesis. The PNIPAm conformation acquired by MD simulation was used to perform docking with IND, PTX, PNIPAm, and water. As expected, binding energy, intermolecular energy, and H-bonding of IND-PNIPAm and PTX-PNIPAm were considerably enhanced (Figure S12). This implied that interactions of IND-PNIPAm and PTX-PNIPAm could be remarkably increased during the process of IND/PNIPAm assembly by modulating PNIPAm to a more desirable conformation, as compared to the initially minimized conformation (Figure S12). After MD simulation, however, the binding energy of PTX-PNIPAm is still lower than that of IND-PNIPAm, indicating that it would be difficult for PTX to form stable assembly with PNIPAm in the absence of IND. Based on these findings, the PTX/IND/PNIPAm complex acquired from MD was further divided into two complexes of PTX/PNIPAm and IND/PNIPAm. Then IND and PTX was separately docked with these complexes as well as PNIPAm conformation acquired from the MD simulation. One could observe that PTX showed strong affinity to the PNIPAm conformation at the site originally occupied by IND in the PTX/IND/PNIPAm complex subjected to the MD simulation (Figure S13 and Figure 6D). Moreover, compared with PTX, IND displayed stronger affinity to PNIPAm at the corresponding site (Figure S13). Actually, when PTX was docked into the IND/PNIPAm complex, the best conformation of PTX preferentially interacted with IND by H-bonding to further strengthen interactions with PNIPAm (Figure S13 and Figure 6E). By contrast, no H-bonding could be observed when PTX was docked into PNIPAm alone. This finding is also coincident with our hypothesis and the MD simulation results. Taken together, the collective effects of multiple interactions among PTX, IND, and PNIPAm contribute to the formation of PTX/IND/PNIPAm assemblies, among which IND serves as a “bridge molecule”. IND may interact with PNIPAm through H-bonding, electrostatic, and hydrophobic forces, while the residual moieties of IND can complex with PTX by H-bonding, π-π stacking, and hydrophobic interactions. Additionally, there are hydrophobic interactions and H-binding between PTX and PNIPAm. As a comparison, we also simulated forces ACS Paragon Plus Environment

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between PTX and PLGA or S100. Consistent with the better PTX loading capability of PTX/IND/PNIPAm assemblies as compared to PLGA or S100 microspheres, PTX exhibited higher binding affinity to IND/PNIPAm than that to PLGA or S100 (Figure S14A). Likewise, the total interaction energy between PTX and PLGA or S100 was dramatically lower than that between PTX and IND/PNIPAm (Figure S14B). Of note, intermolecular forces of PTX/PLGA and PTX/S100 were mainly resulted from hydrophobic force, while both H-bonding and hydrophobic interactions largely contributed to intermolecular forces of PTX, which may cause preferential PTX crystallization when the local drug concentration is high. These findings well agree with the poor loading capacity of PLGA and S100 microspheres for PTX. In Vitro and in Vivo Pharmacokinetic Studies on PTX-Loaded Microspheres. Subsequently, drug delivery performances of assembled PTX/IND/PNIPAm or PTX/SUL/PNIPAm microspheres were further investigated. PTX-containing PLGA or S100 microspheres, with comparable size (Figure S15), were employed as controls. In vitro release of PTX/IND/PNIPAm or PTX/SUL/PNIPAm assemblies in buffer solutions simulating GI conditions demonstrated remarkably enhanced drug dissolution, when compared with PTX-loaded PLGA or S100 microspheres (Figure 5H). Whereas the cumulative release percentage was less than 2% for all microparticles at pH 1.2 within the first two hours, about 84% of total PTX was released within 24 h for PTX/IND/PNIPAm and PTX/SUL/PNIPAm microspheres when the release buffer was switched into PBS with pH 7.4. By contrast, ~54% of total PTX was released from PLGA or S100 microspheres at 24 h. Of note, S100-based microspheres only displayed slightly higher release rate as compared to those based on PLGA. For both IND and SUL, their water solubility may be dramatically enhanced when the pH value of buffer solutions was higher than their pKa of about 4.5.54, 55 Accordingly, the pH-responsive release of PTX from assemblies at pH 7.4 could be attributed to the rapid dissolution of CBC molecules that would result in rapid erosion or disintegration of microspheres. This is in line with in vitro release profiles of IND/PNIPAm assemblies illustrated in Figure 3A-B. Also, the release profiles of PTX are in accordance with molecular simulation results. As indicated in Figure 5I, PTX remained a stable conformation by binding with PLGA in PTX/PLGA complex in PBS. By contrast, with the ionization of IND or S100, significant disassociation of PTX from PTX/IND/PNIPAm or PTX/S100 complexes could be observed. This molecular simulation is also consistent ACS Paragon Plus Environment

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with the fact that assemblies are formed in water while disassembly occurs at pH 7.4. Together, these results demonstrated that assembled microspheres could selectively release PTX in the intestine, and they displayed better pH-triggered release performance as compared to PTX-loaded PLGA or S100 microspheres. Then in vivo pharmacokinetic study was carried out after oral administration of various PTX microparticles in SD rats at the same dose of 10 mg/kg of PTX. Consistent with in vitro release profiles, the assembled PTX/IND/PNIPAm and PTX/SUL/PNIPAm microspheres exhibited significantly higher plasma PTX concentrations in comparison to PTX/PLGA or PTX/S100 microspheres (Figure 7A). Calculation based on these results revealed that PTX/IND/PNIPAm and PTX/SUL/PNIPAm microspheres had significantly larger AUC values than that of PTX/PLGA or PTX/S100 microspheres (Figure 7B). The AUC value of PNIPAm/IND/PTX and PNIPAm/SUL/PTX assemblies was about 1.6 and 1.5 folds of that corresponding to microspheres based on either PLGA or S100, respectively. As for PTX/S100 microspheres, the AUC was only slightly higher than that of PTX/PLGA microspheres (110.1 ± 2.0 versus 105.8 ± 3.4 µg/(L*h)). Since the low bioavailability is one of crucial factors hindering the oral administration of PTX for tumor therapy,61 our assembled PTX microspheres that may partly address this issue are promising for clinical translation. In Vivo Evaluations of PTX-Loaded Microspheres for Cancer Therapy. As a preliminary therapeutic study, in vivo antitumor activity of various PTX microparticles was evaluated in nude mice bearing B16F10 murine melanoma after oral gavage every three days at 10 mg/kg of PTX. At day 1 upon the first administration, the average tumor volume in each group was about 300 mm3. Compared with the control group treated with saline, all PTX-loaded microspheres inhibited tumor growth. However, assembled PTX microspheres afforded more potent efficacy as compared to PLGA and S100 microspheres. After 14 days of treatment, the tumor weight of mice treated with PTX/IND/PNIPAm or PTX/SUL/PNIPAm microspheres was significantly reduced compared with that of mice administered with PTX/PLGA or PTX/S100 microspheres (Figure 7C). This further corroborated the effectiveness of assembled particulate delivery systems. During the whole treatment, we did not find abnormal changes in the body weight of mice treated with PLGA or assembled microspheres (Figure 7D). By contrast, the final body weight of mice administered with PTX/S100 microspheres was significantly decreased as compared to the model control. In line with this, severe ACS Paragon Plus Environment

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inflammation and evident local necrosis could be clearly observed in gastric mucosa and submucosa of the H&E stained sections in the case of PTX/S100 microspheres-treated mice (Figure 7E). Although S100 is a FDA approved expicient for enteric coating in different oral drug delivery systems, its maximal dose for oral applications in human is about 35.24 mg/day, as provided by the FDA database.62 This dose corresponds to about 5.35 mg/kg when it is translated to mice. However, due to the extremely low drug loading content in PTX-loaded S100 microspheres, an extraordinarily high dose of about 749 mg/kg of S100 was administered in mice to reach a PTX dose of 10 mg/kg in this study. Accordingly, this extremely high dose of S100 should be responsible for the observed side effects. Nevertheless, no obvious abnormalities were visible in the intestinal tissue of the PTX/S100 group. Notably, the GI tissues resected from other groups were also normal (Figure 7E). The plasma levels of typical biomarkers including ALT, AST, UREA, and CREA, which are related to liver and kidney functions, showed no abnormal increment (Figure 7F-G). Additionally, we did not find evident injuries or abnormalities in H&E sections of major organs including heart, liver, spleen, lung, and kidney (Figure 7H). Together, these results demonstrated superior efficacy and good safety profile of the newly assembled PTX/IND/PNIPAm and PTX/SUL/PNIPAm microspheres for oral delivery of hydrophobic drugs for tumor therapy. In Vivo Toxicological Evaluations of PNIPAm for Oral Administration. Although PNIPAm and its copolymers have been widely studied for drug delivery,29 gene therapy,56 and tissue engineering,57 few in vivo experiments have been carried out so far to address their safety issue.63 Since above findings revealed that assembled PNIPAm microspheres are highly promising for intestine-targeted drug delivery, herein preliminary tests in mice were performed to interrogate in vivo safety profile of PNIPAm. After a single oral administration of PNIPAm in mice at doses varying from 1.25, 2.5, 5.0, 7.5, to 10 g/kg, no animal death or signs/symptoms of toxicity appeared. Monitoring on the body weight during treatment indicated gradual body weight gain for all mice, and there were insignificant differences between treated and control groups (Figure 8A). Examination on H&E stained pathological sections of major organs excised at day 14 showed indistinguishable injuries for all PNIPAm-treated mice (Figure 8B). Accordingly, PNIPAm showed no evident acute toxicity after oral delivery, even at the dose as high as 10 g/kg, and this dose approaches or even higher than that for many food additives. ACS Paragon Plus Environment

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CONCLUSIONS In summary, by multiple noncovalent interactions-mediated host-guest assembly of CBC and PNIPAm, drugs bearing carboxyl could be highly efficiently loaded into microspheres. Also, carboxyl-deficient hydrophobic drugs could be effectively packaged into this type of host-guest assembled CBC/PNIPAm microspheres. These microspheres, with extremely high drug loading capability as well as desirable redispersibility and scalability, exhibited excellent pH-sensitivity. By selectively releasing cargo molecules under the intestinal conditions, these microspheres could significantly improve the oral bioavailability of loaded therapeutic molecules, which in turn afforded superior therapeutic outcome in rodent models of both inflammation and tumor. Since preliminary in vivo tests demonstrated PNIPAm was safe for oral administration, in combination with the availability of numerous CBCs that have already been widely used in the clinic, these therapeutic assemblies are promising for translation. As PNIPAm with well-tailored molecular weight and delicate structure can be easily synthesized and commercialized by sophisticated polymer chemistry and engineering, the low production cost is another advantage for future applications of these assembled microspheres. Moreover, the current study may provide new perspective in the design and creation of effective materials for oral delivery of a plethora of hydrophobic drugs against chronic inflammation, cancer, and other chronic diseases.

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Scheme 1. Illustration of the host-guest assembly strategy to create pH-responsive microspheres for intestine-targeted drug delivery. CBC, Carboxyl-bearing compound; PNIPAm, Poly(N-isopropylacrylamide).

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A

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M N O P Figure 1. Assembly of CBC/PNIPAm microspheres. (A-C) SEM images of raw IND (A) and dialyzed IND crystals using DMSO as a common solvent (B, C). The image (C) is a high resolution image of (B). (D-I) SEM micrographs showing IND/PNIPAm microspheres at drug/polymer weight ratios of 0.2:1 (D), 0.6:1 (E), 0.8:1 (F), 1:1 (G), 2:1 (H), and 3:1 (I). (J, K) Typical TEM (J) and CLSM (K) images of IND/PNIPAm microspheres at the feeding ratio of 2:1. (L) A typical CLSM image of assembled IND/PNIPAm microspheres based on fluorescein-labeled PNIPAm. (M, N) SEM images of SUL/PNIPAm microspheres assembled at drug/polymer weight ratios of 2:1 (M) and 3:1 (N). (O, P) SEM micrographs of UDCA/PNIPAm microspheres assembled at feeding ratios of 2:1 and 3:1, respectively. ACS Paragon Plus Environment

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Figure 2. Characterization of assembled IND/PNIPAm microspheres. (A) The effect of IND feeding on size of IND/PNIPAm assemblies. (B) Effects of drug feedings on the drug loading content and entrapment efficiency. Data in (A-B) are mean ± S.D. (n = 3). (C) XRD patterns of various samples. The weight ratio of IND/PNIPAm was 3:1 for both assembled microspheres and physical mixture of IND/PNIPAm. (D) DSC curves of IND/PNIPAm assemblies at various weight ratios.

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E Figure 4. Therapeutic performance of assembled IND/PNIPAm microspheres. (A) Therapeutic activity of IND microparticles against acute inflammation (the left panel) and adjuvant-induced arthritis (the right panel) in rats. Data are mean ± S.D. (n = 6). In the acute model, oral administration was performed 0.5 h after paw edema was induced by i.d. injection of carrageen solution in the right-hind foot pad. As for the chronic model, one week after induction of chronic inflammation by i.d. injection of CFA, different IND microspheres were administered by oral gavage every three days. (B) The body weight of rats post various treatments in both acute and chronic inflammation models. Data are mean ± S.D. (n = 6). *p < 0.05 compared with the saline control. (C) H&E stained pathological sections of gastric and proximal small intestinal tissues from rats with acute or chronic inflammation treated by IND/PNIPAm assemblies at 5.0 mg/kg of IND. (D, E) H&E stained sections of organs resected post therapy from SD rats with acute paw edema induced by carrageen (D) or chronic arthritis induced by CFA (E). The control group was treated with normal saline, while both IND/S100 and IND/PLGA groups were treated with IND-containing microspheres at 5.0 mg/kg. For rats treated with IND/PNIPAm assemblies, the corresponding dose was 1.25, 2.5, and 5.0 mg/kg of IND for the low, moderate, and high dose, respectively.

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I Figure 5. Effective loading of PTX into IND/PNIPAm assemblies and in vitro release. (A-D) SEM images of dialyzed PTX (A) and PTX/IND/PNIPAm microspheres assembled at PTX/IND/PNIPAm weight ratios of 0.5:2:1 (B), 1:2:1 (C), and 2:2:1 (D), respectively. (E) The average size of PTX/IND/PNIPAm microspheres. (F) The effects of drug feedings on the loading content and loading efficiency of PTX in IND/PNIPAm assemblies. (G) DSC curves of raw PTX and PTX/IND/PNIPAm assemblies. The assemblies were produced at a weight ratio 2:2:1 for PTX/IND/PNIPAm. (H) In vitro release behaviors of various PTX microparticles in buffer solutions simulating GI pH conditions. The release was performed at pH 1.2 within the first two hours, and then the release medium was switched into 0.01 M PBS at pH 7.4. (I) Molecular modeling of PTX release from microspheres of PTX/IND/PNIPAm, PTX/S100, and PTX/PLGA in PBS. The green and dark cyan molecules represent IND and PTX, respectively. For images in (E, F, and H), data are mean ± S.D. (n = 3).

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E Figure 6. Molecular modeling of PTX/IND/PNIPAm assembly. (A, B) 2D (A) and 3D (B) conformations with the lowest energy for different combinations including IND/PTX, PTX/PNIPAm, and IND/PNIPAm. (C) 2D and 3D conformations showing PTX/IND/PNIPAm complex after a MD simulation. (D) 3D conformations illustrating PTX or IND separately docked in various complex pairs of IND-PNIPAm or PTX-PNIPAm. (E) 2D and 3D conformations of PTX docked in the IND/PNIPAm complex acquired from a MD simulation. In all images, the color of gray, red, and dark blue represents carbon, oxygen, and nitrogen atoms, respectively. The light gray indicates the polar hydrogen atoms that may form H-bonding.

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H Figure 7. Improved oral bioavailability and therapeutic performance of PTX loaded in CBC/PNIPAm microspheres. (A, B) Plasma concentrations of PTX (A) and AUC values (B) of various PTX-loaded microspheres after oral administration in SD rats at 10 mg/kg of PTX. Data are mean ± S.D. (n = 5). (C) The weight of tumors excised from mice bearing melanoma tumors 14 days after treatment with different PTX microspheres by oral gavage at 10 mg/kg of PTX every four days. Data are mean ± S.D. (n = 6). *p < 0.01 and **p < 0.001 versus the model control; ^p < 0.05 versus PTX/PLGA; and +p < 0.05 versus PTX/S100. (D) Changes in the body weight of mice during the treatment. Data are mean ± S.D. (n = 6). *p < 0.05 versus the model control. (E) H&E stained pathological sections of gastric (the upper panel) and proximal small intestinal (the lower panel) tissues from mice post therapy. (F, G) Typical biochemical markers relevant to liver (F) and kidney (G) functions. In all images, the control group was treated with saline alone. ALT, alanine aminotransferase; AST, aspartate aminotransferase; UREA, blood urea nitrogen; and CREA, creatinine. Data are mean ± S.D. (n = 6). (H) H&E sections of main organs isolated from mice after various treatments.

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ASSOCIATED CONTENT

Supporting Information Available. Additional results of Tables 1-3 and Figures S1-15, this material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *Prof. Jianxiang Zhang Email: *[email protected]; [email protected] Department of Pharmaceutics, College of Pharmacy, Third Military Medical University, 30 Gaotanyan Main St, Chongqing 400038, China *Prof. Xiaohui Li Email: *[email protected] Institute of Materia Medica, College of Pharmacy, Third Military Medical University, Chongqing 400038, China Author Contributions ‡

These authors contributed equally (X.Z. and Y.Z.).

Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS We gratefully acknowledge financial support by the National Natural Science Foundation of China (No. 81271695 & 81471774) and the Research Foundation of Third Military Medical University (No. 2014XJY04). This study was also supported by the Program for New Century Excellent Talents in University (No. NCET-13-0703) to JXZ. ACS Paragon Plus Environment

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