Floating molecularly imprinted polymers based on liquid crystalline and polyhedral oligomeric silsesquioxanes for capecitabine sustained release

Accepted Manuscript Floating molecularly imprinted polymers based on liquid crystalline and polyhedral oligomeric silsesquioxanes for capecitabine sustained release Chun-E Mo, Mei-Hong Chai, Li-Ping Zhang, Rui-Xue Ran, Yan-Ping Huang, Zhao-Sheng Liu PII: DOI: Reference:

S0378-5173(18)30988-8 https://doi.org/10.1016/j.ijpharm.2018.12.070 IJP 18039

To appear in:

International Journal of Pharmaceutics

Received Date: Revised Date: Accepted Date:

30 September 2018 26 November 2018 13 December 2018

Please cite this article as: C-E. Mo, M-H. Chai, L-P. Zhang, R-X. Ran, Y-P. Huang, Z-S. Liu, Floating molecularly imprinted polymers based on liquid crystalline and polyhedral oligomeric silsesquioxanes for capecitabine sustained release, International Journal of Pharmaceutics (2018), doi: https://doi.org/10.1016/j.ijpharm.2018.12.070

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Floating molecularly imprinted polymers based on liquid crystalline and polyhedral oligomeric silsesquioxanes for capecitabine sustained release

5

Chun-E Mo, Mei-Hong Chai, Li-Ping Zhang, Rui-Xue Ran, Yan-Ping Huang*, Zhao-Sheng Liu*

Tianjin Key Laboratory on Technologies Enabling Development of Clinical 10

Therapeutics and Diagnostics (Theranostics), School of Pharmacy, Tianjin Medical University, Tianjin 300070, China

Correspondence: Dr. Yan-Ping Huang E-mail: [email protected] 15

Correspondence: Dr. Zhao-Sheng Liu Fax: +086-022-23536746 E-mail: [email protected]

Keywords: molecularly imprinted polymers; liquid crystalline; polyhedral oligomeric 20

silsesquioxanes; capecitabine; sustained release

1

Abstract 25

Molecularly imprinted polymers (MIPs) have drawn extensive attention as carriers on drug delivery. However, most of MIPs suffer from insufficient drug loading capacity, burst release of drugs and/or low bioavailability. To solve the issues, this study designed an imprinted material with superior floating nature for oral drug 30

delivery system of capecitabine (CAP) rationally. The MIPs was synthesized in the presence of 4-methylphenyl dicyclohexyl ethylene (liquid crystalline, LC) and polyhedral oligomeric silsesquioxanes (POSS) via polymerization reaction. The LCPOSS MIPs had extended release of the template molecules over 14.3 h with entrapment efficiency of 20.53%, diffusion coefficient of 2.83×10-11 cm2 s-1, and

35

diffusion exponent of 0.84. Pharmacokinetic studies further revealed the prolong release and high relative bioavailability of CAP in vivo of rats, showing the effective floating effect of the LC-POSS MIPs. The in vivo images revealed visually that the gastroretentive time of the LC-POSS MIPs was longer than non-LC-POSS imprinted polymers. The physical characteristics of the polymers were also characterized by

40

nitrogen adsorption experiment, scanning electron microscopy, thermogravimetric analysis and differential scanning calorimetry analysis. As a conclusion, the LC-POSS MIPs can be used as an eligible CAP carrier and might hold great potential in clinical applications

for

sustained

2

release

drug.

Introduction 45

Capecitabine (CAP) is a new generation of oral fluorouracil analogues that can treat diversified tumors including metastatic breast (Locatelli, et al., 2017) and colorectal cancer (Lampropouls, et al., 2017). As a prodrug, it is metabolized to its active cytotoxic compound: 5-fluorouracil in vivo, and then restrains DNA synthesis. However, after oral administration, CAP tablets could be quickly absorbed and then

50

converted to 5-FU (approximately 60 min for tmax of CAP) (Meulenaar, et al., 2014; Jacobs, et al., 2016). Subsequently, 5-FU is cleared rapidly and almost undetectable in plasma after 6 h. As a result, oral dosing of CAP twice daily, the dosing schedule recommended, is undesirable clinically. Up to now, the most frequently used methods for extended-release of CAP mainly includes the use of new materials such as

55

nanospheres (Liu, et al., 2014), nanoparticles-embedded microcapsules (Liu, et al., 2013), and synergistic self-assembling prodrug (Ma, et al., 2017). The main puzzles associated with these carriers of CAP are the burst release or higher toxicity. One of the methods for settling the issue is to design drug delivery systems (DDS) through which CAP is extended-release. However, developing a satisfactory form for oral

60

drug delivery is a major challenge. Gastric-resident and gastric-retentive drug delivery systems (GRDDS) can provide an extended gastro-intestinal (GI) residence as well as a drug release independent of patient related variables (Davis, 1968). Drug-related problems, i.e., low intestinal solubility, short half-life, or narrow absorption window may be

65

overcome by GRDDS (Strusi, et al., 2008). Due to the ability to maximize drug concentrations within gastric mucosa, this delivery type can provide better local 3

treatment. Floatation, one of the main gastro-retention approaches, was frequently studied to prolong the gastric residence time (GRT) (Singh, et al., 2000). In consideration of the buoyant in the stomach that can not affect the gastric emptying 70

rate, floating drug delivery system (FDDS) has been developed with a bulk density lower than that of gastric fluids (ρ ≈ 1 g-1 cm3) for an increase in GRT (Ishak, 2015). To generate the system of low density, the inclusion of substances or agents in the dosage forms is used to obtain the buoyancy. However, the methods of preparing FDDS are often expensive and cumbersome.

75

One of the cheap alternatives for FDDS is molecularly imprinted polymers (MIPs), which is carrier that is analogous artificial receptors with tailored binding sites for molecular recognition (Liu, et al., 2017; Li, et al., 2013; Pan, et al., 2018). Briefly, a self-assembled prepolymer of functional monomer and template molecule is introduced into the mixture of monomer and crosslinking agent dissolved in one or

80

more solvents, and then a three-dimensional polymer matrix is formed (SmolinskaKempisty, et al., 2017; Li, et al., 2017). After the polymerization reaction is completed, the template is removed from the prepared polymer, and the sites of specific recognition are left in the polymer matrix complementary to the shape, size and chemical functional group of the original template molecule (Aşır, et al., 2017).

85

The prepared MIPs have a lot of merits, e.g., structure-activity predetermination (Liu, et al., 2014), high selectivity (Jenkins, et al., 2012), practicability (Ashley, et al., 2018; Sun, et al., 2018), simple preparation process and low cost (Li, et al., 2018). Up to now, the MIPs have ranges of successful applications in the field of solid phase extraction (Peng, et al., 2011; Speltini, et al., 2017), chromatographic separation 4

90

(Guerreiro, et al., 2008), drug delivery (Zaidi, et al., 2016; Upadhyay, et al., 2018) and so on. By mimicking the interactions between natural receptors and target molecules, MIPs are capable of retaining desired drugs based on their reciprocal interaction, enhancing the loading capacity and enabling sustained drug release. MIPs had

95

attracted broad interest in DDS as very exciting and useful drug delivery vehicles, e.g., magnetic MIPs against doxorubicin for triggered cancer therapy (Griffete, et al., 2015), MIPs against aminoglutethimide for zero-order sustained release (Tang, et al., 2015), and MIPs against metronidazole for pH-triggered drug delivery (Marcelo, et al., 2018). However, CAP is a small molecule with flexible

100

conformations, and therefore, to obtain molecular imprinting against CAP is a hard work. As a result, rationally designing specific MIP materials with extended-release is necessary. Recently, an approach to MIP preparation as DDS using cooperative effect of liquid crystalline (LC) and polyhedral oligomeric silsesquioxanes (POSS) has been

105

developed (Zhang, et al., 2018). LC, known as physical crossing monomer with the unique mesogenic groups, can be used to prepare excellent MIPs with much higher capacity because of low chemical crosslinking degree of the resulting LC MIP thus higher accessibility to the sites than conventional MIPs (Liu, et al., 2013; Zhang, et al., 2015). POSS are nanoscale cage-shaped structure with sizes of 1-3 nm and are

110

believed to be the smallest particles of silica. Using selectively designed POSS blocks to construct nanocomposites offers a powerful tool to control arrangement at the

5

nanoscale and tailor novel properties by their macroscopic organization (Pu, et al., 2013; Cai, et al., 2016; Zhu, et al., 2017). By use of LC and POSS simultaneously in the preparation of MIPs, the resultant seductive functional materials can improve the 115

ability of specific recognition of the MIPs further and extend release for template/target molecules (Zhang, et al., 2018). The observations are particularly valuable in the situation where the target molecule has difficult to be imprinted. Additionally, the good floatation of the LC POSS MIP can give rise to superior release for oral drug delivery with excellent relative bioavailability.

120

Herein, we have developed LC-POSS MIPs against CAP for floating oral drug delivery (Fig. 1). Methacrylic acid (MAA) was used as functional monomers, a series of LC and POSS monomer as co-monomer to fabricate the CAP-imprinted polymers in the presence of ethylene dimethacrylate (EDMA). The synergistic effect of dual monomers of LC and POSS is expected to contribute greatly to the performances of

125

the LC-POSS MIPs and high bioavailability in vivo. Furthermore, gastroretentive images in vivo and pharmacokinetic studies of CAP in rats were performed on the LCPOSS MIPs. 2. Materials and experimental methods 2.1. Materials

130

Capecitabine (CAP, 98%) and 2,2-azobis (2-isobutyronitrile) (AIBN, AR grade) were obtained from J&K Chemical Co. Ltd. (Beijing, China). Polyhedral oligomeric silsesquioxane (POSS) (MA 0702, AR grade) was from Hybrid Plastics Reagent Co. Ltd. (Hunan, China). Ethylene glycol dimethacrylate (EDMA, 98%) were purchased

6

from Sigma-Aldrich (St. Louis, MO, USA). Liquid crystalline monomer, 4135

methylphenyl dicyclohexyl ethylene (MPDE, 98%) and all other liquid-crystalline monomers of 4-methylphenyl dicyclohexyl butylene (MPDB, 98%), 4-myanophenyl cyclohexyl ethylene(CPCE, 98%), 4-cyanophenyl cyclohexyl propylene (CPCP, 98%), as well as 3,4-difluorophenyl dicyclohexyl butylene (DFDB, 98%), was obtained from Hebei Maison Chemical Co. Ltd. (Hebei, China). Methacrylic acid

140

(MAA, AR grade) was obtained from Beijing Donghuan Chemical Reagents (Beijing, China). Rhodamine B (RhB, AR grade) was provided by Beijing Dingguo Changsheng Biotechnology Co.Ltd. Other analytical reagents were supplied by Tianjin Guangfu Technology Development Co. Ltd. (Tianjin, China) and Tianjin Chemical Reagent Co. Ltd. (Tianjin, China).

145

2.2 Preparation of LC and POSS-based MIPs In this work, the LC monomer and POSS-based MIPs (LC-POSS MIPs) were prepared by bulk polymerization. The formulations of polymers preparation are display in Table S1. General procedure of MIP synthesis can be summarized in the following steps: Firstly, CAP (template), MPDE (physical crosslinker), POSS (co-

150

monomer), AIBN (initiator) were dissolved in toluene and acetonitrile (porogens), after sonication in a 20 mL glass ampoule for 10 min. Afterwards, MAA (functional monomer) and EDMA (chemical crosslinker) were added into the mixture above and sonicated for 10 min again to ensure all materials dissolved. Then dissolved oxygen was removed by purging with nitrogen for more than 5 min. After that, the

155

prepolymerization mixture was placed in a water bath at 53°C for 4 h. After the 7

reaction, excessive POSS was removed from the polymers by Sechelt extractor with THF for 12 h, then washed with methanol-acetic acid (9:1, v/v) for 36 h until CAP could not be detected by UV-visible spectrophotometer at a wavelength of 310 nm. NIPs were synthesized in the same manner but without template CAP in the 160

formulation. Other three controls, POSS LC-free MIPs, LC POSS-free MIPs, conventional MIPs without POSS and LC monomer were synthesized in similar way. 2.3 Molecular modeling Molecular modeling was undertaken to optimize the MIP preparation procedure. The ratio between the template molecule and the functional monomer was performed

165

by molecular modeling software program Hyperchem 7.5 (Hypercube Inc., Gainsville, FL). Firstly, the optimization of the template and the monomer complex was obtained by the molecular mechanics (MM+), and then the semi-empirical mechanics algorithm was used for further conformational optimization. Finally, the lowest energy of the conformation was calculated with an ab initio (3–21 G) quantum

170

mechanic basis set (Farrington et al., 2015). The binding energies between template and monomer were calculated by the Eq. (1)

∆𝐸 = [𝐸𝑐𝑜𝑚𝑝𝑙𝑒𝑥 ‒ 𝐸𝐶𝐴𝑃 ‒ 𝐸𝑀𝐴𝐴]

(1)

2.4 Equilibrium adsorption experiment To evaluate the adsorption capacities of MIP, CAP solutions (3 mL, 0–5 mM) 175

were added to centrifuge tube containing 10.0 mg of the dry polymers respectively. After shaken at room temperature for 5 h, the resulting mixtures were centrifuged at 10,000 rpm for 10 min. Then the CAP concentration in the supernatant was measured

8

after dilution using UV-vis spectrophotometer at the wavelength of 310 nm. The amount of CAP bounded to the polymers at equilibrium was calculated using the 180

following Eq. (2)(Tang et al., 2015): Q  e

(C - C )  V 0 e M

(2)

Where Qe (mmol g-1) is the binding amount of polymers; C0 and Ce (mmol L-1) are the initial and equilibrium concentrations of CAP in the solution, respectively; V (L) is the volume of CAP solution. M (g) is the weight of the polymers. 185

The Langmuir model was also used to evaluate the binding capacities of MIPs based on follow Eq. (3)(García-Calzón and Díaz-García, 2007): Qe 

Q max KCe 1  KCe

(3)

where Qmax is the amount of CAP adsorbed for monolayer saturation capacity (mmol g-1), and K is a Langmuir constant (L mmol-1). 190

Imprinting effect of MIPs is evaluated by imprinting factor (IF), which is defined as Eq. (4)(Tang et al., 2015):

IF 

QMIP QNIP

(4)

Where QMIP and QNIP are the maximum amounts of CAP bounded on MIP and NIP (control), respectively. 195

2.5 Drug release experiments in vitro 2.5.1 Optimization of loading concentration 40 mg LC-POSS MIPs (M3) and LC-POSS NIPs (N3) were soaked in 4 ml ethanol containing 6, 8, or 10 mg mL-1 of CAP for 3 days. Subsequently, the samples 9

were centrifuged, then the concentration of the supernatant was measured by an UV200

vis spectrophotometer to calculate the drug loading amount. After that, the polymers were dried under room temperature. 30 mg of dry polymer loaded with CAP was placed into a dialysis bag with 2 ml of ethanol solution, then dipped the dialysis bag into 100 ml ethanol solution. The absorbance of the release drug was measured by UV-vis spectrophotometry at appropriate time interval.

205

2.5.2 Effect of different polymers on CAP loaded and release ability Under optimal loaded conditions, the LC-POSS MIPs, LC MIPs, POSS MIPs, MIPs and their corresponding NIPs were loaded with CAP of the same concentration above. The release experiments were carried out as the same method. The amount of drug loaded, percentage of the drug loaded, encapsulation efficiency was calculated

210

using the following equation, respectively (Tang et al., 2015): Amount of drug loaded 

Percent drug loaded 

(5)

Weight of the loaded drug  100% Total weight of durg and polymers

(6)

Weight of the loaded drug  100% Weight of durg

(7)

Entrapment efficiency 

215

Weight of the loaded drug Weight of polymers

2.5.3 Mathematical analysis of release kinetics for CAP The diffusion mechanism of CAP from polymer to diffusion medium can be analyzed by diffusion coefficient D using the short-time approximation equation (8) (Ritger, et al., 1987),

10

M

220

t  4 ( DE t )0.5 (8) 2 M   Where Mt/M represents the diffusion percentage of drug, D is the relevant

diffusional coefficient, δ is the diffusional distance and t is the diffusion time. Korsmeyer-Peppas equation (Ritger et al., 1987) was used to fit the data of CAP release: Mt  kt n M 225

(9)

Here, k represents a constant, n is the diffusional exponent which is an indication of diffusional release mechanism. 2.6. Water absorption capacity of the polymers Accurately

pre-weighed

dry

polymers

were

mixed

in

excess

ultrapure water respectively and shocked for three days. Then the excess water was 230

removed by filtration. Weights of the swollen polymers were measured. The water absorption capacity was usually expressed as swelling ratio which evaluated by equation (10)(Bai et al. 2015). The experiments were carried out in duplicate.

W -W WS  t 0 W 0

(10)

Here, Wt and W0 is mass of the swollen polymers and dried polymers 235

respectively. 2.7 In vitro cytotoxicity Cell viability was assessed by 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-diphenytetrazoliumromide (MTT) assay. MCF-7 cells were seeded in a 96-well plate

11

(1×105 cells/well). Polymers with a rage of proper concentrations were added to the 240

cell cultures for 24 h, and then 20 µL of the MTT solution (5 mg mL-1) was added to each well of 96-well plates and incubated for 4 h. After incubation, 100 μL DMSO was added to dissolve the insoluble formazan crystals. The absorbance was measured at 490 nm using the Multimode Reader (Model 1680, Bio-Rad, America). The experiment was conducted in triplicate and results were expressed as mean ± standard

245

deviation. The cell viability was calculated as follows: IR % = (OD with drug / OD without drug ) × 100. 2.8 Floating behavior The buoyancy of polymers was observed in different solvent, water and PBS solution (pH 7.4).

250

2.9 Gastroretentive images of polymers in vivo Gastroretentive imagines of polymers in vivo was to analysis the time that polymers retained in the stomachs of the rats. 160 mg of the LC-POSS MIPs (M3), LC MIPs, POSS MIPs, MIPs loaded with CAP was absorbed with 640 mg fluorescence of Rhodamine B, respectively, and stirred with the speed of 50 rpm

255

under 4℃ for 10 h. After that, washed away the fluorescence adsorbed on the surface of polymers and dried in vacuum at room temperature and avoided light at the same time. 5 mg of the labeled polymers was dispersed separately in 2.0 mL simulated gastric juice, and the healthy male Wistar rats were administrated by gavage. After

260

that, the rats were euthanized in every half an hour. Then their stomachs were 12

removed and imaged on the IVIS Spectrum imaging system (Caliper Life Science, Hopkinton, MA, USA), based on the excitation filter set of 570 nm and emission of 620 nm. 2.10 In vivo pharmacokinetic studies in rats 265

Healthy male Wistar rats weighting 180–200 g (8–10 weeks old) were provided by Experimental Animal Centre of Academy of Military Sciences PLA China (Tianjin, China). All Wistar rats were raised by the Department of Laboratory Animal Science of Tianjin Medical University (laboratory animal certificate: syxk2014-0004). The experiments were controlled by the local ethics committee and conducted in

270

accordance with the Laboratory Animal Management Rules of the People’s Republic of China and regulations of Tianjin Municipality Implementation of the Regulations on Laboratory Animal Management Rules. The LC-POSS MIPs (M3), LC-POSS NIPs (N3), MIPs loaded with CAP and a commercial tablet of CAP were dispersed in 2.0 mL of physiological saline,

275

respectively,

then

administrated

intragastrically

(1

mg

kg-1

CAP).

After

administration, 100 μL of blood sample was collected from the orbital vein of the rats at a specified time interval into a heparinized centrifuge tube, centrifuged at 4000 rpm for 5 minutes, and then 100 μL of the supernatant was transferred into the 100 μL of methanol and followed by centrifugation at 13,000 rpm for 5 minutes. Finally, the 280

organic layer was placed in a clean tube, and stored at -20°C after drying. The samples were dissolved in the mobile phase and then separated by HPLC system at 310 nm using C18 reverse phase column (100-5C18, 250 × 4.6 mm;

13

Kromasil, Sweden). The column temperature was 25℃ and flow rate was 1.0 mL min1.

285

Mobile phase was acetonitrile: 0.1% acetic acid solution (27:73, v/v). The detection

limit was 10.0 ng mL-1 of CAP. To evaluate the absorption and metabolism of CAP released by the polymers in rats, the pharmacokinetic parameters, Tmax and Cmax, were measured. Tmax and Cmax could be directly obtained using actual observations, and AUC0-12 was calculated by linear trapezoidal method. The relative bioavailability (F) was evaluated by the ratio

290

of the polymer AUC to the commercial tablet AUC. 3. Results and discussion 3.1 Characterization of LC POSS imprinted polymers 3.1.1 Scanning electron micrograph and FTIR spectral studies Fig. 2 shows the scanning electron micrograph of the polymer particles. The LC-

295

POSS MIPs and NIPs consisted of coagulum, while the conventional MIPs and NIPs showed microparticles. The LC-POSS polymers showed relatively smooth and glassy, different from non-LC-POSS polymers. The aggregates of the LC-POSS MIPs illustrated the much uniform structure of the LC-POSS NIPs. The results suggested that the introduction of POSS and LC monomer as well as the proportion had

300

significant effect on the morphology of the polymers. A dramatic shift in the polymerization rate may be responsible to the different structure of LC POSS and LCPOSS-free polymers. Drug molecule before and after remove from polymers and the affiliation of LC or POSS monomer were analyzed by FTIR spectrum. In Fig. S1, CAP showed

14

305

characteristic bands at 3522 and 3220 cm−1 due to O-H/N-H stretching vibrations. The bands of 1647 cm-1, 1688 and 1718 cm−1 were attributed to pyrimidine carbonyl stretching vibrations, urethane carbonyl stretching vibrations, respectively. A downward shift of N-H stretching vibrations to 3447 cm−1, pyrimidine carbonyl stretching vibrations to 1637 cm-1 were seen for the LC-POSS MIPs/CAP, indicating

310

the presence of hydrogen bonding between CAP and MAA. Compared to the LCPOSS MIPs/CAP, the LC-POSS MIPs and LC-POSS NIPs had no characteristic bands of CAP. It was observed that C-H stretching vibrations in 3030 cm-1 appeared in LC while weaken in LC MIPs due to the broken of vinyl groups. For POSS and the LC-POSS-based polymers, all showed Si-O stretching vibrations to 1100 cm−1.

315

3.1.2 Nitrogen adsorption and desorption The pore size distribution and surface areas of the polymers was characterized with nitrogen adsorption-desorption. As shown in Fig. 3, the polymers showed the similar Type IV isotherm, which was associated with mesoporous structures. Type H3 hysteresis loop of the polymers was indicative of the aggregates of plate-like particles

320

leading to slit-shaped pores (Sing, et al., 1985). What’s more, the desorption curves were above the correspondingly adsorption curves, indicating the existence of only little amount of micropores (Yuan, et al., 2008). The narrow pore diameter distribution and low average pore diameter of the polymers displayed in the mesoporous region about 4-6 nm was significantly prominent (Fig. S2), suggesting

325

that the size of the pores formed in the matrices may play a key role of binding sites. As observed in Table 1, the BET surface area of the LC-POSS MIPs (4.26 m2

15

g−1) was about 5 times larger than that of the LC-POSS NIPs (0.83 m2 g−1), and the pore volume (0.012−3 cm3 g−1) was larger than the latter (0.007−3 cm3 g−1), which indicated that the distinct adsorption property of the LC-POSS MIPs can be attributed 330

to the imprinting effect. Similarly, the LC-POSS MIPs had larger BET surface area than those MIPs that were made without LC or POSS while the BET surface area of the conventional MIPs and corresponding NIPs was almost equal. However, the pore diameter of LC-POSS MIPs was smaller than other polymers. In contrast, conventional MIPs had larger pore volume and pore diameter but smaller surface

335

area. This was in consistent with the observations of Paul et al. (Paul, et al., 2017) and Yang et al. (Yang, et al., 2018) that smaller particle sizes would produce polymer matrices with larger surface area. To some extent, it indicated that the addition of LC and POSS monomer into the polymerization system reduced the pore size of the resulting LC-POSS MIPs.

340

3.1.3 Thermal characteristics of the polymers To determine the thermal characteristics of the obtained polymers, the materials were tested using TG analysis and DSC. As shown in Fig. 4a, the LC-POSS MIPs presented a smooth TG curve with maximum residual mass of 17.9% as the quality of the samples no longer changed. The temperature was the highest when half of the

345

weight was lost. Additionally, the curves of the MIPs and NIPs were similar, in which the weight of the samples left was 0.6%. This improvement of thermodynamic property can be attributed to the creation of rigid structure of the polymers as a result of the combination of MPDE and POSS.

16

DSC thermograms indicated that pure LC and POSS monomer had a sharp 350

endothermic peak at low temperature of 70℃ and 112℃, respectively, representing their crystalline nature (Fig. 4b). The LC-POSS MIPs and other polymers showed similar change in the phase temperature characteristics at approximately 400°C. All these results illustrated that the thermodynamic stability of the LC-POSS MIPs was greatly enhanced associated with the combination of LC and POSS monomer to the

355

polymer with favorable rigid structure. 3.2 Effect of polymerization parameters on imprinting To evaluate imprinting effect, the adsorptive ability of the MIPs and corresponding NIPs was investigated. Fig. 5 shows that higher amount of CAP is absorbed on the LC-POSS MIPs than the LC-POSS NIPs with a higher imprinting

360

factor of 4.6. The large difference of adsorption ability between the LC-POSS MIPs and NIPs revealed the presence of imprinted sites which was dependent on the noncovalently binding of CAP and MAA was critical for adsorption. In contrast, the conventional MIPs and MIPs prepared with LC or POSS monomer, had little difference in adsorption of imprinted and non-imprinted polymers, and the imprinting

365

factors of the LC MIPs, POSS MIPs, as well as conventional MIPs were 2.0, 1.5, and 1.2, respectively. Obviously, the imprinting factor of the LC POSS MIPs was about two fold increase than the conventional MIPs prepared without LC and POSS monomer. 3.2.1 Template to functional monomer ratio

370

The ratio of template to functional monomer (T/F) could significantly affect the

17

quality and quantity of the imprinted site. The binding energy between CAP and MAA was calculated using molecular modeling software. ∆Ecomplex was -31.64, 57.06, and -67.27 kcal/mol when the ratio of CAP to MAA was 1: 4, 1: 5, 1: 6, respectively. The complex in ratio of 1:7 can not be formed because the ∆E was 375

positive. Therefore, the different molar ratio of MAA to CAP was used to prepare LCPOSS MIPs (M9, 1:4, M3, 1:5, and M10, 1:6) by adjusting CAP amount added while other parameters in pre-polymerization mixture were kept as constant (Fig. S3a). When the T/F ratio was 1:4, there was little difference in absorbance amount of CAP between the imprinted and non-imprinted polymers (M9, IF = 1.1). Because of the

380

excessive content of the template in this case, the selectivity of imprinted polymers against CAP was smaller due to unfavorable equilibrium for the formation of CAPMAA complex. When T/F ratio was decreased, there was enough functional monomer that the equilibrium may be moved toward complex formation and higher yield of good sites can be produced (Yilmaz, et al., 1999). Herein, the optimum T/F ratio was

385

1:5 with imprinting factor of 4.6. When the ratio was decreased to 1:6 further, the imprinting factor (IF = 1.2) was reduced in spite of higher than the T/F ratio of 1:4. The suggests indicated that for LC-POSS MIP, the convention calculation of binding energy is invalid to formula selection for MIP preparation and spatial factors may play a crucial role in affinity.

390

3.2.2 Effect of nature of LC monomer In this experiment, the LC monomers used were composed of aromatic or aliphatic rigid LC groups, which contain cyclic structures and alkyl chains with

18

terminal unsaturated ethylene bonds (Fig. 6). The alkyl chain whose units usually varied from 2 to 6 coupling with polymer backbone and might lead to a change of the 395

crosslinking density of the network and affect the mass transfer of template molecule (Mitchell, et al., 1997; Binet, et al., 2007). To explore the effect of LC structure on the imprinting, five LC monomer with different structure, including MPDE, MPDB, CPCE, CPCP and DFDB were used to investigate the selective recognition ability of CAP. As showed in Fig. S3b, the LC-POSS MIPs made with MPDE had excellent

400

recognition ability towards CAP. However, the adsorption amounts of the MIPs prepared with other LC monomers were all lower and no obvious regularity was observed. By a comparison, it can be concluded that greater rigidity of liquid crystalline and increasing the number in liquid crystalline is favorable to keep the memory to the template. What’s more, the effect of the substituent in the fluorine

405

atom of the benzene ring was minor in exerting its influence on the LC-POSS MIPs. 3.2.3 Ratio of POSS to LC monomer Previous finding suggested that an enhancement of the hydrophobic nature and the rigidity of the functional monomers may increase the potential in molecular recognition of the resulting MIP (Zhang et. al. 2018). Thus, the molar ratio of POSS

410

to LC monomer may also play a critical factor of influencing the adsorption ability of MIP. In the present study, we changed the molar ratio of POSS to LC monomer (0, 1.2:2.7, 2.4:1.5, and 3:0.9) by keeping the total molars of POSS and LC monomer a constant. Meanwhile, the content of EDMA in the polymerization system was not varied. As shown in Fig. S3c, the influence of molar ratio of POSS to LC monomer on

19

415

the polymer adsorption capacity was obvious, and the greatest adsorptive capacity was obtained when the ratio of POSS to LC was 3:0.9 (IF = 4.3). The imprinting factor and adsorption amount were increased when the amount of POSS was larger, which might be due to the increased hydrophobic nature and rigidity as the ratio of POSS in the resulting MIP.

420

3.3 In vitro release studies 3.3.1 Optimum concentration of drug loading Prior to the release studies in vivo, it was necessary to find the optimal loading concentration of CAP. We tried to use HCl pH 1.0, PBS pH 7.0, acetonitrile and acetic acetate buffer pH 7.0 (9:1), 0.1% sodium dodecyl sulfate as release medium.

425

However, the polymers may float in the buffer and drug release can not be measured, thus ethanol was selected as the medium (Wang et. al. 2018). As a result, the release behavior of CAP of the polymers in ethanol solution was investigated since all the polymers can be dispersed in the solution. M3, the most effective LC-POSS MIPs, was soaked in the CAP ethanol solution at three different concentrations for three

430

days before use. The changes in the release behaviors of CAP due to the modification of the loaded concentration were observed (Fig. 7). It’s worth noting that barely initial burst effects were observed at the concentration of 8 mg mL-1, indicating the excellent controlled release due to the stronger binding CAP at the imprinted site. When the drug loading was 10 mg ml-1, 48% of CAP was released in 2 hours. It may be due to

435

the too large drug concentration, causing more non-selective adsorption of CAP on the surface of the polymer. Therefore, at higher loading of CAP, the non-controlled

20

release observed was determined by the conventional adsorption rather than imprinting effect, thus the release rate was faster. It also showed that the larger the initial drug concentration was, the longer release time would be among the three 440

loaded concentration but not significantly. Interestingly, the cumulative release of LCPOSS MIPs was around 80% while the LC-POSS NIPs arrived at 100% when the polymers were soaked with 8 mg mL-1 of CAP solution, which concentration was smaller than other loaded concentration. Furthermore, as shown in Table S3, the diffusional coefficients (DE) of the LC-POSS MIPs were 4.25, 2.83, and 5.80 when

445

the loaded CAP concentration was 6 mg ml-1, 8 mg ml-1, and 10 mg ml-1, respectively. It indicated that the LC-POSS MIPs had slower release kinetics than other concentration at a soaking concentration of 8 mg mL-1. In order to probe release mechanism, Korsmeyer-Peppas equation was used to fit the data obtained. Good linearity ranging from 0.948 to 0.996 was achieved. When

450

the loaded concentrations were 6 mg mL-1, 8 mg mL-1 and 10 mg mL-1, respectively (Table S3), the diffusional exponents (n) of CAP were 0.71, 0.83 and 0.62, respectively, suggesting non-Fickian release of the drug. This diffusion mechanism meant that the diffusion of the drug from the polymers and polymer relaxation were comparable. The release rate at the different loaded concentration may be influenced

455

for two reasons. The first can be explained by the driving force and cross-link density for the drug diffusion (Denizli et al., 1988). When loaded with 10 mg mL-1, more CAP may be adsorbed on the surface of the polymers than that loaded with 8 mg mL1.

Thus, stronger driving force and increased swelling ability with decreased cross-

21

link density all result in fast diffusion of the drug. When loaded with low 460

concentration, diffusion coefficients may be depended on the concentration gradient, while high concentration will slow down the diffusion, i.e., loading of 8 mg mL-1 was slow than 6 mg mL-1 (Singh et al., 2011). When the concentrations of CAP loaded was 8 mg mL-1, the release profile was close to zero-order release which had the capacity to release sustained amount of CAP in a constant manner during an extended

465

time period, resulting in improved patient therapeutic efficacy and compliance (Tieppo et al., 2012, Wang et al., 2016). The difference generated by different loaded concentration was attributed to more or less recognition site on the LC-POSS MIPs. The optimal concentration of CAP loading (8 mg mL-1) was dependent on the maximum amount of the imprinted sites, which resulted in an optimum character of

470

the release. 3.3.2 Evaluation of loading ability and diffusional coefficients of polymers On the basis of the above results, we assessed the loading ability and release profile of CAP in vitro from the LC-POSS MIPs, LC MIPs, POSS MIPs, MIPs and the corresponding NIPs after loaded with 8 mg mL-1 CAP ethanol solution. As shown

475

in Table 2, the LC-POSS MIPs displayed the highest value in the amount of drug loaded, percent drug loaded and encapsulation efficiency. Moreover, the LC MIPs and POSS MIPs were also superior to the conventional MIPs on the basis of the amount of the drug loaded, percent drug loaded and encapsulation efficiency. It demonstrated that as the introduction of LC and POSS monomer into the synthesis system, the high

480

amount of drug loaded, percent of drug loading and encapsulation efficiency were

22

obtained. Furthermore, the drug loading of POSS alone was 30 mg g-1, which is attributed to the size of POSS unit in the range of 1.2 to 1.5 nm, around 10 times than that of CAP. This can be explained that the reason why the drug loading of the polymers containing POSS was higher than that without POSS, and the polymers with 485

LC and POSS were significantly higher than that of the other control groups. Since POSS can physically encapsulate CAP, the drug loading of the LC-POSS NIPs were high, while the greater drug loading capacity of the LC-POSS MIPs may be attributed to the imprinting accompanied with physical encapsulation (Yang et al., 2016). The LC-POSS MIPs showed the longest release time, up to 13.4 h (Table 2).

490

Additionally, the value of the diffusional coefficients (DE) for the LC-POSS MIPs, LC MIPs, POSS MIPs as well as MIPs were 2.83, 6.03, 31.02, 3.71 cm2 s-1, smaller than their corresponding non-imprinting polymer matrixes, respectively. As shown in Table S4, LC-POSS MIPs showed the smallest diffusion coefficient. The release profiles of CAP from the LC-POSS MIPs and LC-POSS NIPs were remarkable

495

different (Fig. S3), while the controls LC MIPs, POSS MIPs, and MIPs showed a smaller difference than the corresponding NIPs. Two reasons can account for the behaviors of sustained release: one is that the diffusion characteristics of the polymer affect the release process, and the other is the interaction between the template and the imprinted polymer. Further, the synergistic effect of two factors had may work to

500

accomplish with sustained release (Chen et al., 2018). 3.4. Floating behavior of different polymers The LC-POSS MIP and POSS MIP showed outstanding floating properties in

23

water (Fig. 8), indicating the promising devices as GDDS. When they were placed in water, the LC-POSS MIP and POSS MIP floated directly, and remained buoyant at 505

least 24 hours. In contrast, the LC-MIP and conventional MIP samples sank to the bottom of water. Here, it is the first time to find the floating behavior of POSS-based MIP in the aqueous medium. Different floating behavior of the LC-MIP with previous reports (Zhang et al., 2017) may be attributed to less amount of LC monomer in the polymer (13% mol).

510

3.6 Preliminary safety evaluation The unloaded and loaded polymers were examined for their toxicity against MCF-7. As shown in Fig. 9, the lowest cell viability was all > 80% for the unloaded LC-POSS MIPs, LC-POSS NIPs, LC MIPs, POSS MIPs and MIPs in different concentrations, suggesting that these polymers had not any cytotoxicity to MCF-7

515

cells. Cell viability decreased slightly when the CAP loading was 10 μg mL-1 on the polymers, but still higher than 80%, since CAP was pro-drug which exhibits low toxicity in the present form. The result leads to the conclusion that the devices studied here can be used as non-toxic delivery matrix. 3.5 Gastroretentive time of drug in LC-POSS MIPs

520

To evaluate floating of the sustained drug delivery devices studied here, the gastroretentive effect of different polymers was investigated. Before intragastric administration, we soaked the Rhodamine B-labeled polymers in the simulated gastric juice for 5 hours and found that the fluorescence intensity of the polymers did not change much within 3 hours. This was in consistent with our previous results (Zhang

24

525

et al., 2017), indicating that this method can be used to label the polymers for subsequent experiments. Gastric images of the rats within a certain period of time are shown in Fig. 10. Here, the decrease or disappearance of the fluorescence signal reflected the drug absorption or gastric emptying. Additionally, the fluorescent intensity was

530

significantly reduced after intragastric administration of 1.5 hours. It could be seen that after 0.5 hours of administration, the fluorescence intensity of the drug in the stomachs of the rat were the strongest. The fluorescence intensity of all drugs in the stomachs decreased with time, indicating the absorption process. After 1.5 hours, the fluorescence intensity of the conventional MIPs was very weak, and almost no

535

fluorescent substance was detected by 2 hours. The LC MIPs and POSS MIPs were not detected after 2.5 hours, while the intensity of the LC-POSS MIPs was stronger than the other control groups in 2 hours. As shown in the images, the LC-POSS MIPs retained in the stomachs longer than the LC MIPs, POSS MIPs as well as traditional MIPs. The signal intensity of the LC-POSS MIPs disappeared until 2.5 hours,

540

indicating that the LC POSS MIPs had an enhanced effect of floating, which could improve the ability of drug release of the LC POSS MIPs materials and achieve better sustained release. Water absorption capacity of the LC-POSS MIPs, POSS MIPs, LC MIPs, MIPs were 0.058, 0.066, 1.21, 1.15 g/g, respectively, indicating that the higher water

545

absorption capacity can be obtained on the better floatability of polymers. The disparity of the results might be due to the differences in polymer structures, such as

25

degree of crosslinking, hydrophilic-hydrophobic interactions, etc. (Bajpai et al., 2016). Due to the greater water absorption capacity of the non-POSS MIPs, water can penetrate the outer polymers and the network swelled, leading to formation and 550

amplification of diffusion apertures just like hydrogel. In contrast, the space chains for the LC-POSS MIPs,was more stable, so sustained release CAP can be achieved. 3.7 In vivo pharmacokinetic study To evaluate the sustained release effect further, CAP pharmacokinetic experiments of the LC-POSS MIPs were investigated, and the results were compared

555

to that of the LC-POSS NIPs, LC MIPs, POSS MIPs, MIPs, and commercial tables. After the polymers were loaded with the CAP concentration same as in vitro, intragastric administration to the rats was performed with the equivalent dose of 1.0 mg kg-1. The comparison of plasma concentration-time curves and pharmacokinetic parameters were depicted in Fig. 11 and Table 3, respectively. The maximum plasma

560

concentration (Cmax) and the area under the curve (AUC0–12) of CAP from the LCPOSS MIPs, LC-POSS NIPs, LC MIPs, POSS MIPs, MIPs and commercial tables were 36.1, 33.8, 36.8, 30.8, 21.1, 32.5 ng mL-1 and 162.3, 77.8, 98.2, 75.8, 44.3, 96.1 ng mL-1 h, respectively. The results suggested that the relatively high amount of CAP released from the LC-POSS MIPs in rats’ body circulation. Furthermore, the LC-

565

POSS MIPs had the longest time (3 hours) to reach the maximum plasma concentration (tmax), while LC-POSS NIPs, LC MIPs, POSS MIPs, MIPs, had shorter tmax of 0.5 hour or 1 hour. Additionally, the LC-POSS MIPs maintained a rational blood concentration between 0.5 and 8 hours after gastric irrigation of the loaded LC-

26

POSS MIPs and extended release for up to 12 hours. However, CAP from the 570

commercial tables could not be detected in plasma after 6-8 hours, which was in line with the previous studies (Budman, et al., 1998; Jacobs, et al., 2016). Compared with the commercial tables, the LC-POSS MIPs had a prominent bioavailability of 168.9%, more than 1.6 times that of other polymers, 1.5 times than the previous study (Singh, et al., 2015).

575

4. Conclusions We have successfully prepared MIPs with the incorporation of LC and POSS monomer for sustained release of CAP. The cooperative effect of LC and POSS led to successful imprinting to the hard target molecule, CAP. The LC-POSS MIPs displayed remarkable floating behavior and longer gastric residence time in the

580

aqueous medium than the non-floating MIP. Compared with the commercial tablet, the relative bioavailability of the gastro-floating LC-POSS MIPs loaded CAP was 168.9%. Collectively, the rationally formulated LC-POSS MIPs may shed new light on the sustained release of CAP and also hold great potential for anti-cancers carrier. Acknowledgments

585

This work was supported by National Natural Science Foundation of China (Grant No.

21775109).

Compliance with ethical standards Conflict of interest The authors declare that they have no competing interests.

27

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Legends Fig. 1 Schematic representation of LC-POSS MIPs preparation. Fig. 2 Scanning electron microscopy (SEM) images of LC-POSS MIPs (M3), LC755

POSS NIPs (N3), MIPs (M13), NIPs (N13). Fig. 3 Nitrogen adsorption–desorption isotherms of LC-POSS MIPs, LC-POSS NIPs, MIPs and NIPs. Fig. 4 Thegravimetric analysis results (a) and differential scanning calorimeter analysis results (b).

760

Fig. 5 Effect of liquid crystal and POSS on the absorptive capacity. Each data point represents the mean ± standard deviation (n = 3). Fig. 6 Structure of LC monomer used in present study. Fig. 7 Release profiles in vitro of CAP from LC-POSS MIPs and LC-POSS NIPs soaked in three different concentrations. Each data point represents the mean ±

765

standard deviation (n = 3). Fig. 8 Floating of LC-POSS MIPs, LC MIPs, POSS MIPs, and conventional MIPs in aqueous medium. Fig. 9 Cytotoxicity assay of the unloaded polymers (a), CAP and loaded polymers (b). Fig. 10 Fluorescent images of the polymers in the stomachs of rats (n = 3).

770

Fig. 11 Plasma concentration–time curves of the loaded LC-POSS MIPs, LC-POSS NIPs, LC MIPs, POSS MIPs, MIPs and the commercial tablet of CAP. Mean values with error bars of standard deviation (n = 3) are plotted.

36

Table 1 Pore properties of LC-POSS MIPs, LC-POSS NIPs, MIPs, NIPs. polymers LC-POSS MIPs LC-POSS NIPs MIPs NIPs

SBET (m2 g−1) 4.26 0.83 3.41 3.46

St -3 (10 m2

g−1)

9.81 1.15 3.67 3.98

Vp (cm3 g−1) 0.012 0.007 0.085 0.015

Dmean (nm) 5.01 26.70 92.61 15.31

775

Table 2 Evaluation of capecitabine loading of different polymers. Amount of drug loaded (mg g-1) 164.21 130.84 44.53 31.88 60.64 67.55 25.27 36.43

Polymers LC-POSS MIPs LC-POSS NIPs LC MIPs LC NIPs POSS MIPs POSS NIPs MIPs NIPs

Percent drug loaded (%) 9.12 7.27 2.47 1.77 3.37 3.75 1.40 2.03

Entrapment efficiency (%) 20.53 16.36 5.57 3.98 7.58 8.44 3.16 4.56

Time to final release (h) 13.42 9.17 13.30 13.30 12.08 9.08 12.30 12.30

780

Table 3 Pharmacokinetic parameters of capecitabine in rats. Samples LC-POSS MIPs LC-POSS NIPs LC MIPs POSS MIPs MIPs CAP

Tmax (h) 3.0 0.5 1 0.5 0.5 0.5

Cmax (ng mL-1) 36.1 33.8 36.8 30.8 21.1 32.5

37

AUC0-12 (ng mL-1 h) 162.3 77.8 98.2 75.8 44.3 96.1

F (%) 168.9 81.0 102.2 78.8 46.1

785

38

790

39

40

795

41