Thymopentin Nanoparticles Engineered with High Loading Efficiency

Mar 18, 2014 - method.19 TP5 and SPC (1:30 w/w) were dissolved in dimethyl sulfoxide .... In the following seven days, the rats in group 3 were given ...
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Thymopentin Nanoparticles Engineered with High Loading Efficiency, Improved Pharmacokinetic Properties, and Enhanced Immunostimulating Effect Using Soybean Phospholipid and PHBHHx Polymer Chengyu Wu,† Mengtian Zhang,† Zhirong Zhang,† Ka-Wai Wan,‡ Waqar Ahmed,‡ David A. Phoenix,‡ Abdelbary M. A. Elhissi,*,‡ and Xun Sun*,† †

Key Laboratory of Drug Targeting and Drug Delivery Systems, Ministry of Education, West China School of Pharmacy, Sichuan University, Chengdu 610041, P. R. China ‡ Institute of Nanotechnology and Bioengineering, School of Pharmacy and Biomedical Sciences, University of Central Lancashire, Preston, United Kingdom ABSTRACT: Formulation of protein and peptide drugs with sustained release properties is crucial to enhance their therapeutic effect and minimize administration frequency. In this study, immunomodulating polymeric systems were designed by manufacturing PHBHHx nanoparticles (NPs) containing thymopentin (TP5). The release profile of the drug was studied over a period of 7 days. The PHBHHx NPs containing TP5-phospholipid (PLC) complex (TP5-PLC) displayed a spherical shape with a mean size, zeta potential, and encapsulation efficiency of 238.9 nm, −32.0 mV, and 72.81%, respectively. The cytotoxicity results showed the PHBHHx NPs had a relatively low toxicity in vitro. TP5 entrapped in the NPs could hardly release in vitro, while the NPs had longer than 7 days release duration after a single subcutaneous injection in Wistar rats. The immunodepression rat model was built to evaluate the immunomodulating effects of TP5-PLCNPs in vivo. The results of T-lymphocyte subsets (CD3+, CD4+, CD8+, and CD4+/CD8+ ratio) analysis and superoxide dismutase (SOD) values suggested that TP5-PLC-NPs had stronger immunoregulation effects than TP5 solution. In conclusion, an applicable approach to markedly enhancing the loading of a water-soluble peptide into a hydrophobic polymer matrix has been introduced. Thus, TP5-PLC-NPs are promising nanomedicine systems for sustained release effects of TP5. KEYWORDS: PHBHHx, TP5, NPs, controlled release, phospholipid complex, immunoregulation

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rapid burst effect, 6−8 which could waste considerable proportions of the drug during preparation and can often lead to side effects after drug administration. Moreover, microparticles are difficult to sterilize and can cause serious pain to the patient when given as a subcutaneous injection since thick needles are needed for the administration of microparticle dispersions.9 By contrast, nanoparticles (NPs) are less painful to the patient when parenterally injected, and following their manufacture, they can be aseptically filtered or prepared under aseptic conditions; these characteristics make NPs better candidate formulations for TP5. However, on the basis of literature findings, the currently available TP5 delivery systems based on solid lipid, chitosan, or PLGA nanoparticles still need

INTRODUCTION

Controlled release of water-soluble peptides and proteins has drawn great interests for decades. Thymopentin (TP5) is a synthetic water-soluble pentapeptide (water solubility > 1 g/ mL) corresponding to the active site of the thymopoietin. TP5 has been widely used in clinic including primary and secondary immune deficiency, infections, rheumatoid arthritis, cancers, and autoimmune diseases.1−3 However, the duration of TP5 treatment usually lasts for one week to six months, and the plasma half-life of this peptide is very short (about 30 s).4 Thus, designing a single injection of TP5 that can have a therapeutic effect for prolonged periods is highly desirable to maximize patient compliance and potentially reduce therapy expenses. Meanwhile, it has been reported that the potency of TP5 can be enhanced by slow infusion in vivo.5 Therefore, a sustained release system is needed to reduce the injection frequency and increase the therapeutic effect. Microparticles are usually used to prolong the duration of drug release. However, for water-soluble peptides, microparticles may have relatively low encapsulation efficiency with © XXXX American Chemical Society

Special Issue: Recent Molecular Pharmaceutical Development in China Received: December 3, 2013 Revised: March 3, 2014 Accepted: March 18, 2014

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Preparation of TP5-PLC Loaded PHBHHx Nanoparticles (TP5-PLC-NPs). TP5-PLC-NPs were prepared by emulsion−solvent evaporation.20 The lyophilized TP5-PLC (1:30, w/w) and PHBHHx were dissolved in chloroform with different weight ratios to constitute the organic phase. F68 (0.5% w/v) and DOC-Na (0.5% w/v) were dissolved in deionized water to constitute the aqueous phase. The organic phase was added into the aqueous phase at a volume ratio of 1:10, followed by probe-sonication at 400 W for 10 times each was for 3 s with 5 s rest intervals in between the sonication cycles to avoid overheating of the preparation. The resultant white emulsion was stored under vacuum at 37 °C for at least 15 min to remove the chloroform. TP5-PLC-NPs containing PEG2500 (10%, weight ratio) were prepared using the same method by adding PEG2500 in chloroform. Characterization of TP5-PLC-NPs. The average diameter and polydispersity index (PDI) of the TP5-PLC-NPs were determined using a laser light scattering instrument (Zetasizer Nano ZS90, Malvern Instruments Ltd., U.K.) at 25 °C. Zeta potential (ZP) of the NPs was analyzed via electrophoretic mobility using the same instrument following the selection of the relevant software option. The morphology of TP5-PLCNPs was viewed using a conventional scanning electron microscope (SEM, JSM-5900LV, JEOL, Japan) at an accelerating voltage of 5 kV. Encapsulation Efficiency (EE) of TP5. The encapsulation efficiency (EE) of TP5 was measured by using an ultrafiltration centrifugation method. TP5 loaded in 1 mL of TP5-PLC-NPs was extracted using 5% (w/v) Triton X-100 solution with aid of sonication. The resultant suspension was added into the Millipore filter (MWCO = 3500 Da), and the filtrate was injected into HPLC to measure the total amount of TP5 originally loaded into the NPs. Another 1 mL of TP5-PLC-NPs was added into the Millipore (MWCO = 3500 Da) without using Triton X-100 and sonication. The obtained filtrate was also injected into HPLC to measure the amount of free TP5 (Wf). Therefore, the encapsulation efficiency of TP5-PLC-NPs was calculated using the following equation:

to be administered everyday to maintain therapeutic concentrations of the drug in the blood circulation.10−12 Thus, development of a sustained TP5 NPs would be highly advantageous to reduce the injection frequency and increase the therapeutic effect of the drug. Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx), one of the most promising biodegradable semicrystalline aliphatic polyesters of the polyhydroxyalkanoate (PHA) family, has been widely studied due to its unique properties, including full anaerobic degradability, high biocompatibility, safety, and absence of toxicity and immunogenicity of their degradation products.13−18 In our previous studies, we found that insulin incorporated into PHBHHx nanoparticles were able to maintain the hypoglycemic effect for 3 days after subcutaneous release.19 In this study, for the first time a complex consisting of TP5 peptide and phospholipid was developed and incorporated into PHBHHx NPs. We investigated the release behavior of TP5 in vivo through measuring the FAM-TP5 concentration in blood. Also, in order to study the therapeutic effect of TP5 NPs, an immunodepression model using Wistar rats was established, and superoxide dismutase (SOD) value and CD3+, CD4+, and CD8+ T cells were measured.

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EXPERIMENTAL SECTION Materials. Soybean PC (Lipoid S-100) was purchased from Lipoid GmBH (Ludwigshafen, Germany). TP5 and FAM-TP5 were a gift of Chengdu Kaijie Biotechnologies Co. Ltd. PHBHHx (MW = 534 000) containing 11 mol % of R-3hydroxyhexanoate (HHx) was kindly donated by Guo Qiang Chen (Qinghua University, China.). Poloxamer 188 (F68) was purchased from BASF, Germany. Sodium deoxycholate (DOCNa) was supplied by Amresco (Solon, USA). Cyclophosphamide was provided by Sigma (St. Louis, USA). SOD kit was obtained from Nanjing Jiancheng Bioengineering Institute (Jiangsu, China). Antirat CD3 APC and Antirat CD8b PE were purchased from eBioscience (San Diego, CA, USA). Antirat CD4 FITC was supplied by Biolegend (San Diego, CA, USA). All other chemical reagents were of analytical grade. Preparation of Thymopentin Phospheolipid Complex (TP5-PLC). TP5-PLC was prepared by adapting our previous method.19 TP5 and SPC (1:30 w/w) were dissolved in dimethyl sulfoxide (DMSO) containing 5% (v/v) of glacial acetic acid followed by mixing in a horizontal shaker at 37 °C for 2 h. The resultant solution was freeze-dried using a freezedryer (Thermo Savant ModulyoD-230, USA) for 24 h. The lyophilized TP5-PLC was sealed and stored at 4 °C before conducting further studies. Solubility Study of TP5-PLC. The solubility of TP5-PLC was determined in chloroform by adding TP5-PLC (1:10, w/ w) into chloroform in a sealed glass container. The container was gently shaken on an orbital shaker at 25 °C for 2 h. The sample was then filtered using a vacuum pump and 0.45 μm organic membrane filters under the fume cupboard. The filtrate was withdrawn and diluted before analysis by high performance liquid chromatography (HPLC). Meanwhile the solubility of free TP5 in chloroform was measured using the same method. Determination of Interaction between TP5 and SPC. Fourier transform infrared spectrophotometry (FT-IR Spectrometer, BRUKER VECTOR 22, Germany) was used to study the interaction between TP5 and SPC. The IR spectra of TP5, SPC, the TP5-SPC complex, and a physical mixture of TP5 and SPC were obtained by using the KBr method.

EE(%) = (Wt − Wf )/Wt × 100%

(1)

Preparation and Evaluation of Freeze-Dried TP5-PLCNPs. In order to remove chloroform completely and store NPs properly, TP5-PLC-NPs were freeze-dried, and to prevent aggregation upon rehydration, 10% (w/v) trehalose was added as a cryoprotectant prior to freeze-drying. The NPs suspension with trehalose was frozen at −45 °C overnight and subsequently placed in the freeze-drier for 24 h. The freezedried samples were sealed and stored at room temperature. All subsequent experiments were conducted using rehydrated freeze-dried samples. The NPs before and after freeze-drying were characterized. The lyophilized TP5-PLC-NPs were redispersed in distilled water. The mean particle size, PDI, ZP, and encapsulation efficiency of the resuspended NPs were measured by the methods described earlier. In Vitro Release Studies. For a standard in vitro release test, 2 mL of TP5-PLC-NPs, TP5-PLC-NPs containing PEG2500, or TP5 solution was placed into a dialysis bag (MWCO = 3500 Da), which was immersed into 10 mL phosphate buffer saline solution (PBS, 0.01M, pH 7.4) containing 0.02% NaN3 followed by shaking in a horizontal shaker under controlled conditions (200 rpm, 37 °C). At time intervals, samples from the release medium were withdrawn B

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subcutaneous injection only on the fourth day. On the eleventh day, blood samples were collected from the rats and loaded into the anticoagulant tubes for the T-lymphocyte subsets analysis and the SOD activity assay. Measurement of the Lymphocyte Subsets. The lymphocyte subsets were determined by multiparameter flow cytometry with three-color analyses. The immunofluorescent staining of the whole blood was performed by adding 100 μL of the anticoagulant whole blood to a test tube containing preadded fluorescent antibodies (12.5 μL of antirat CD3 APC, 5 μL of antirat CD4 FITC, and 12.5 μL of antirat CD8b PE). After incubation for 20 min at 25 °C in dark, red blood cells were lysed using the hemolysin. The samples were washed thoroughly in PBS by centrifugation at 350 × g for 5 min, and the cell sediments were resuspended in 0.5 mL of PBS. The samples were kept on ice, and the T-lymphocyte subsets were analyzed within 4 h using a flow cytometer (Cytomices FC 500, Beckman coulter, USA). The CD4+/CD8+ ratios were calculated by the amounts of labeled CD3+/CD4+ T cell and CD3+/CD8+ T cell in the blood samples. Measurement of SOD Value. The SOD activity assay was based on the SOD kit instruction, using a UV spectrophotometer (Varian, USA) at 550 nm. For each sample assay, 20 μL of plasma was used, and all the assay validation met the requirements of the SOD kit instruction. Statistical Analysis. The data were expressed as mean ± standard deviation (SD). Statistical analysis was carried out using the Student’s t tests. The difference between two groups was considered to be statistically significant when the p value was less than 0.05.

and replaced with fresh drug-free PBS solution. TP5 concentration in the samples was quantified by HPLC. Cytotoxicity of Blank NPs. The cytotoxicity of the blank NPs was evaluated using an MTT assay. L929 cells were cultured in RPMI-1640 supplemented with 10% FBS, 100 units/mL of penicillin, and 100 mg/mL of streptomycin under 5% CO2 at 37 °C. The cells were seeded in a 96-well plate in 200 μL medium per well at a density of about 1 × 104 cells/well for 24 h. The medium was then replaced with 200 μL of medium-containing NPs at different concentrations and incubated for 24 h. The NP-containing media were then removed to avoid interference in the assays. Then, 0.5 mg/mL MTT solution (20 μL) and medium (200 μL) were added, and cells were incubated for another 4 h. The MTT-containing media were removed and cells were rinsed 3 times with PBS. DMSO (200 μL) was added to lyse the cells followed by incubation at 37 °C for 15 min. MTT absorbance was measured at 570 nm using Varioskan Flash spectral scanning multimode reader (Thermo Scientific, America). The cell viability (%) was calculated according to the formula (Atreated − Abackground)/(Acontrol − Abackground) × 100, while the control cells were not exposed to the samples and the background had no cells. The MTT assay was performed in quintuplicate. In Vivo Studies Using Rats. All 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 Sichuan University (Chengdu, China). Wistar rats (male, body weight 200−220 g) were obtained from the Experimental Animal Center of Sichuan University and fed on a light and dark cycle. All animals were allowed free access to standard rat chow and water. Temperature and relative humidity were kept at 25 °C and 50%, respectively. Pharmacokinetics of 5-FAM-TP5-PLC-NP. The animals were randomly divided into two groups each to receive 5-FAMTP5 aqueous solution (6 mg/kg) and 5-FAM-TP5 NPs (6 mg/ kg) by subcutaneous injection, respectively (n = 5). Blood samples were collected at scheduled time and centrifuged at 4000 rpm for 4 min. Plasma (0.1 mL) was collected and mixed with 0.4 mL of ethanol using vortex mixing for 1 min to extract 5-FAM-TP5. The suspension mixture was centrifuged again at 8000 rpm for 10 min in order to acquire 5-FAM-TP5 solution. The plasma concentration of 5-FAM-TP5 was measured using an RF-5301PC spectrofluorophotometer (Shimadzu, Japan). Drug concentration versus absorbance demonstrated linearity in the range of 2−80 ng/mL (R2 = 0.9996). The pharmacokinetic parameters were calculated based on a noncompartmental model using the DAS version 2.0 program. The Cmax is the maximum plasma concentration of 5-FAMTP5, and the Tmax is the time corresponding to Cmax. The AUC0‑t was calculated using the linear trapezoidal rule. The MRT is the mean retention time of 5-FAM-TP5. Pharmacodynamics of TP5-PLC-NPs. The wistar male rats were equally divided into four groups (n = 5). For three consecutive days, the rats in groups 2, 3, and 4 were given cyclophosphamide solution intraperitoneally at a dose of 35 mg/kg/d to build immunodepression rat models. The rats in groups 1 and 2 without further treatment were used as the normal control and the immunodepression control, respectively. In the following seven days, the rats in group 3 were given TP5 solution (0.6 mg/kg) by tail vein injection every day, and the rats in group 4 received TP5-PLC-NPs (6 mg/kg) by

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RESULTS Solubility Studies. The prepared TP5-PLC was a yellow sticky powder. The solubility of TP5 in chloroform was found to be 16.34 μg/mL, while the solubility of TP5-PLC (1:10 w/ w) in chloroform was much higher (12.84 mg/mL). Moreover, the solubililty of TP5-PLC in chloroform was 786 times higher than that of the free TP5. Thus, the results indicate that TP5PLC is much more lipophilic than PT5 and that the complexation between the peptide and phospholipid was successful. IR Spectra Analysis. The infrared spectra of TP5, SPC, TP5-SPC complex, and TP5 and SPC physical mixture are shown in Figure 1. There was a significant difference between the physical mixture and the complex. The spectrum of the physical mixture showed an additive effect of TP5 and SPC, in

Figure 1. Infrared spectra of (a) TP5, (b) SPC, (c) TP5−SPC physical mixture, and (d) TP5−SPC complex. C

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Table 1. Characterization of TP5-PLC-NPs Prepared with Different Formula

a

formulationa

size (nm)

PDI

ZP (mV)

EE (%)

1:30:30 1:30:60 1:30:90

221.6 ± 3.4 238.9 ± 4.7 246.7 ± 4.0

0.243 ± 0.022 0.181 ± 0.016 0.190 ± 0.017

−34.3 ± 1.4 −32.0 ± 2.3 −31.2 ± 2.2

52.66 ± 4.31 72.81 ± 5.41 57.87 ± 4.42

The formulation represents the weight ratio of TP5/SPC/PHBHHx. Values are mean ± SD (n = 3).

Preparation and Evaluation of Freeze-Dried TP5-PLCNPs. Freeze-drying (lyophilization) was found convenient and effective to remove residual chloroform and improve the stability of TP5 NPs suspensions. The freeze-dried NPs appeared as a white fluffy cake, which was easy to rehydrate in order to form NPs by gentle mixing for 10 s. No significant changes in particle size, zeta potential, and entrapment efficiency were observed before and after freeze-drying. In fact, there was a trend of EE enhancement following freezedrying in which no statistically significant was observed (Table 2). At the same time, the NPs without adding trehalose after freeze-drying became a compact cake, which cannot be suspended into water again. Therefore, 10% (w/v) of trehalose was a suitable cryoprotectant to protect TP5-SPC-NPs during freeze-drying. In Vitro Release of Freeze-Dried NPs. The in vitro release of TP5 was studied using the dynamic dialysis method (Figure 3). In TP5 solution group, the peptide was completely

which the characteristic absorption peaks of TP5 were still present at 1394 and 1652 cm−1. However, in the spectrum of their complex, the two characteristic absorption peaks of TP5 were almost absent. Also, compared with TP5, SPC and physical mixture, the intensity of the absorption peak at 3375 cm−1 was decreased in the spectrum of the complex. Moreover, no new peaks were observed in the mixture and complex. These observations indicated that physical interactions between TP5 and SPC took place, resulting in the formation of a complex between the peptide and phospholipid. Preparation and Characterization of TP5-PLC-NPs Prior to Freeze-Drying. Through a previously established procedure, TP5-based nanomedicines were produced by an emulsion−solvent evaporation method. The mean particle size, PDI, zeta potential, and encapsulation efficiency of NPs containing different PHBHHx were characterized (Table 1). The size increased slightly but significantly (P < 0.05) upon increasing the concentration of PHBHHx. However, PDI has decreased (P < 0.05) when higher concentrations of PHBHHx were used. Zeta potential values were all negative, and higher concentrations of PHBHHx have slightly decreased the charge intensity. Importantly, the encapsulation efficiency of TP5PLC-NPs was at highest (72.81%) when the formulation 1:30:60 was used. Therefore, only the TP5-PLC-NPs with a weight ratio of 1:30:60 were used for subsequent studies as they exhibited the smallest PDI values with the highest entrapment efficiency. The morphology of the NPs was investigated by SEM (Figure 2), which revealed that the NPs have well-defined spherical shapes.

Figure 3. Release profile of TP5 from TP5 NPs, TP5 NPs containing PEG2500, and TP5 solution in vitro (n = 3).

released in 8 h, showing that the dialysis bag did not restraint the release of TP5. For the TP5 NPs, the release profile can be divided into two phases. The first phase represented the release of free drug, which lasted for 8 h (i.e., the burst effect), resulting in the release of 39.27% of TP5 during that period of time. The second release phase started after 8 h and represented the sustained release phase, during which only about 5% of the loaded TP5 was released at a relatively low rate, agreeing with the previous research findings.21 By contrast, in PEG2500 containing TP5 NPs, TP5 was completely released in 8 h. Cytotoxicity of Blank NPs. L929 cells have been recommended by many research investigators as reference cell lines for the cytotoxicity testing of polymers and NPs.22,23 We studied the cytotoxicity of blank NPs on L929 cell proliferation. For blank nanoparticles, the cytotoxicity increased when the concentration of blank NPs was increased. Only when the concentration of blank NPs was increased to 400 μg/mL

Figure 2. SEM image of TP5 phospholipid complex loaded PHBHHx nanoparticles (TP5-PLC-NPs).

Table 2. Characterization of TP5-PLC-NPs before and after Freeze-Drying; Values Are Mean ± SD (n = 3) sample

mean size (nm)

PDI

ZP (mV)

EE (%)

before freeze-drying after freeze-drying

246.9 ± 3.5 259.0 ± 2.0

0.183 ± 0.004 0.261 ± 0.005

−32.00 ± 0.95 −30.47 ± 0.06

68.80 ± 4.72 73.58 ± 2.82

D

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efficacy of TP5 in various formulations. As an immunomodulator, TP5 is an established therapeutic peptide that regulates the CD4+/CD8+ ratio to normal levels.25 Therapeutic effect of TP5-PLC-NPs was evaluated using the immunosuppressive rats. In our study, the values of CD4+/CD8+ in blood of the immunosuppressive rats were higher than that of normal rats (p < 0.01), and the percentage of CD3+ T Cell in the immunosuppressive rats were lower than that of normal rats (Table 4), which indicated the successful establishment of the immunosuppression model. After administration of TP5 solution and TP5-NPs, the values of CD4+/CD8+ decreased significantly (p < 0.05). The CD4+/CD8+ values in TP5-NPs group displayed no statistical difference with the normal group but the TP5 solution group did by demonstrating higher CD4+/CD8+ values, indicating that only the NP-based formulation was capable of reversing the suppression of the immune system. The same phenomenon appeared in the percentage of CD3+ T cell (Table 4). These results demonstrated that TP5-PLC-NPs had a better function on Tlymphocyte subsets than TP5 solution. The changes of the SOD activity in blood could illuminate the immune abnormality of the body, which was chosen to reflect the pharmacodynamic properties of different TP5 formulations.26 As demonstrated in Figure 6, the SOD values of the immunodepression rats were significantly decreased compared with those of the normal control rats, and the SOD levels of the immunodepression rats could be increased after the administration of TP5 NPs or TP5 solution. Moreover, the SOD values in the TP5 NPs group were significantly increased, with no statistical differences when compared to the SOD level in the normal animals group. There were no significant differences between the immunodepression group and TP5 solution group. The results indicated that the TP5 NPs had a stronger pharmacodynamic action than TP5 solution.

cytotoxicity started to be detected (Figure 4), which is evident for the relatively low toxicity of SPC-PHBHHx NPs.

Figure 4. Cytotoxicity assay performed by studying the cell viability using L929 cells exposed to a range of blank SPC-PHBHHx NP concentrations (n = 5).

Pharmacokinetics of 5-FAM-TP5-PLC-NPs. To evaluate the release profile of TP5-PLC-NPs in vivo, the pharmacokinetic studies were carried out using Wistar male rat. Previous studies showed the TP5 was not stable in the plasma, and the half-life was about 30 s in human plasma.4 It is difficult to determine the plasma concentration of TP5 by conventional methods such as HPLC and HPLC−MS. Therefore, TP5 was fluorescently labeled by 5-FAM. As shown in Figure 5, in the 5-FAM-TP5-PLC-NPs group, the plasma 5-FAM concentration decreased slowly and the

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DISCUSSION In our study, in order to achieve a long lasting and sustained release of TP5 from NPs, we selected a highly hydrophobic biodegradable material PHBHHx. As TP5 is a water-soluble peptide, the preparation of TP5 phospholipid complex was necessary to reduce the water-solubility of TP5 (i.e., consequently increase TP5 solubility in chloroform) for the purpose of enhancing the encapsulation of TP5 into PHBHHx NPs. The phospholipid complex was formed by physical interactions such as hydrogen bonding force and van der Waals force between TP5 and SPC. This hypothesis was verified by the IR spectra analysis. Moreover, through the solubilization studies we can see that the solubility of TP5 in chloroform was increased 786 times in the form of phospholipid complex, which could demonstrate evidence for the formation of TP5 phospholipid complex.10 Encapsulation of hydrophilic drugs into hydrophobic nanoparticles is a considerable challenge. The encapsulation efficiency of TP5 in solid lipid nanoparticles prepared by O/ W or W/O/W method was 5.2% and 1.7%, respectively.27 Only 33% of TP5 was incorporated into lectin-conjugated PLGA nanoparticles by double emulsion-solvent evaporation method.10 In our study, higher encapsulation efficiency of TP5 was achieved, being 72.81% upon employing a direct emulsionsolvent evaporation method for the formation of TP5 phospholipid complex, which significantly increased the solubility of TP5 in chloroform.

Figure 5. Mean plasma 5-FAM-TP5 concentration−time curve of each experiment group (n = 5).

fluorescence signal remained in the plasma at day 7 after the administration of 5-FAM-TP5-NPs. In the FAM-TP5 solution group, the concentration of 5-FAM increased to maximum concentration after 2 h, and then decreased rapidly and dropped to undetectable concentrations (i.e., almost zero) after 48 h. Pharmacokinetic parameters are provided in Table 3. The mean retention time (MRT) value of 5-FAM-TP5 NPs was 17.325 h, which was longer than that of the 5-FAM-TP5 solution (8.847 h). Pharmacodynamics of TP5-PLC-NPs. In immunological deficient patients, the values of CD3+CD4+ and CD3+CD8+ Tlymphocyte subsets in blood are usually abnormal with altered CD4+/CD8+ ratio.24 Thus, the ratio of CD4+ and CD8+ Tlymphocyte subsets was used to evaluate the therapeutic Table 3. Pharmacokinetic Parameters of 5-FAM-TP5 Solution and 5-FAM-TP5-NPs (n = 5) parameters

5-FAM-TP5 solution

5-FAM-TP5 NPs

Cmax (μg/mL) Tmax (h) AUC0‑t (μg/mL·h) MRT0‑t (h)

7.758 ± 0.632 2±0 26.811 ± 2.607 8.847 ± 0.996

2.553 ± 0.379 1.2 ± 0.447 15.616 ± 1.003 17.325 ± 2.56 E

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Table 4. Values of T-Lymphocyte Subsets in Blood of Rats (n = 5)

a

group

CD3+ (%)

normal control immunodepression control TP5 solution, i.v. TP5 NPs, s.c.

43.3 0 ± 3.8 19.03 ± 5.1a 25.03 ± 5.0a,b 34.98 ± 7.7b

CD3+CD4+ (%) 65.38 62.88 62.67 57.80

± ± ± ±

5.8 2.4 2.3 9.5

CD3+CD8+ (%) 31.20 13.08 17.83 25.03

± ± ± ±

6.0 2.3a 2.1a 6.0b

CD4+/CD8+ 2.18 4.91 3.54 2.30

± ± ± ±

0.62 0.75a 0.28a,b 0.12b,c

p < 0.05 vs normal control. bp < 0.05 vs immunodepression control. cp < 0.05 vs TP5 solution, i.v.

In our research, an enhanced therapeutic effect of TP5 PHBHHx NPs was found. There is a statistical difference in the T-lymphocyte subsets and SOD value between TP5 PHBHHx NPs group and TP5 solution group. When compared to the study conducted by Wang and co-workers (2006) in which TP5 was administrated at 10 mg/kg/d for 7 consecutive days intranasally,25 the PHBHHx NPs used in the present study showed an equivalent effect of immunoregulation with 6 mg/kg single subcutaneous injection in 7 days. Also, in the experiments conducted by Jin et al., the immunoregulation function of TP5 was found after it was orally delivered at 2 mg/ kg/d for 7 consecutive days.31 In conclusion, a novel lipophilic TP5 phospholipid complex was prepared, and then using a solvent evaporation method, the complex was incorporated into NPs (TP5-PLC-NPs). The mean particle size, spherical shape, and high TP5 encapsulation efficiency suggest that the developed formulation is applicable for parenteral administration. Trehalose (10% w/w) was an efficient cryoprotectant for freeze-drying the TP5-PLC-NPs. The sustained release properties of TP5-PLC-NPs in vitro and in vivo provided evidence for the presence of a strong interaction between TP5 and phospholipid, indicating successful formation of the drug−lipid complex and stability of the NPs. Additionally, the cytotoxicity experiment showed that the blank NPs had a very low toxicity, indicating that the formulation is safe. TP5-PLC-NPs could release TP5 for more than 7 days after a single subcutaneous injection, which displayed better therapeutic effect than TP5 solution injected once a day for 7 consecutive days. Thus, an enhanced therapeutic effect of TP5 formulation with a 7 day release period was made. Meanwhile, TP5-PLC-NPs have a great potential to serve as a sustained release nanomedicine. The patient compliance could potentially be significantly improved by TP5-PLC-NPs due to the improved therapeutic effect and the substantial decrease of the injection frequency.

Figure 6. SOD activity of each experiment group (n = 5). *p < 0.05; **p < 0.01.

In the in vitro release studies, the TP5 was released completely in 8 h in the TP5 solution group. In the TP5 NPs group, the free TP5 was released quickly within 8 h, while the TP5 entrapped in the NPs exhibited a very slow release profile. This indicated that the PHBHHx NPs are relatively firm and that there was an interaction between TP5 and SPC, resulting in controlled release of the drug. By contrast, in the TP5 NPs containing PEG2500 group, although the EE was similar to the TP5 NPs, TP5 was released completely in 48 h. It can be explained that hydrophilic channels may have been formed in the NPs, resulting in rapid disintegration of the structure of NPs. In the pharmacokinetics experiment, it was observed that the fluorescence of 5-FAM could last more than 7 days in the rat plasma. Compared to our previous studies, two reasons may contribute to the two-phase release of TP5-PLC-NPs: First, the weight ratio of TP5 to SPC in phospholipid complex was 1:30, which prevented TP5 release from the NPs. Second, high molecular weight PHBHHx (MW = 534 000) was used to prepare NPs, which prolonged the degradation time of the NPs. In the pharmacodynamics experiments, we did not separate the free TP5 from the NPs suspension. This is because the high dose of TP5 in this study is within the safe dose range, and no serious side effects have been reported even when the dose was elevated to 5000 mg/kg.28,29 Additionally, high concentrations of TP5 can stimulate the immune system further, leading to a better therapeutic effect.30 In order to achieve the same therapeutic effect, we give a relatively high concentration of TP5 solution (0.6 mg/kg) in the positive group. Also, intravenous injection provides the most potent of TP5 and is applied in clinic,30 so in the TP5 solution group, TP5 solution was injected through the vein. In the pharmacodynamics experiment, the NPs were given once a week for two reasons; first, according to the pharmacokinetics experiment results, the fluorescence of 5FAM disappeared on the eighth day in the rat plasma, while the half-life of 5-FAM may be 12 h at the low concentration. This means that the TP5 can be released from the NPs for at least 7 days. Second, since the therapeutic dosage of TP5 is very low, 0.38 μg/kg/d is sufficient to establish a positive effect, and the potency of TP-5 can be enhanced in vivo by slow infusion.5 The pharmacodynamics results confirmed that our dosing frequency choice was appropriate.

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AUTHOR INFORMATION

Corresponding Authors

*(A.M.A.E.) Tel: +44 01772 89 5807. E-mail: aelhissi@uclan. ac.uk or [email protected]. *(X.S.) Tel: +86 28 85502307. Fax: +86 28 85501615. E-mail: [email protected]. Author Contributions

All authors contributed equally to this work. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful for the financial support from the University of Central Lancashire, United Kingdom and the National Basic Research Program of China (973 program, No: 2013CB932504). We also thank Mr. Jianbo Li and Dr. Qiang Peng for their help in the present study. F

dx.doi.org/10.1021/mp400722r | Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Molecular Pharmaceutics

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Article

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ABBREVIATION TP5, thymopentin; TP5-PLC, thymopentin phospholipid complex; PHBHHx, poly(3-hydroxybutyrate-co-3-hydroxyhexanoate); TP5-PLC-NPs, thymopentin phospholipid complex nanoparticles; NPs, nanoparticles; SEM, scanning electron microscope

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dx.doi.org/10.1021/mp400722r | Mol. Pharmaceutics XXXX, XXX, XXX−XXX