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Formulation and evaluation of poly(lactic-co-glycolic acid) microspheres loaded with an altered collagen type II peptide for the treatment of rheumatoid arthritis a

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Jintian He , Huiqi Li , Chao Liu , Gaizhen Wang , Lan Ge , Shufen Ma , Lijing Huang , b

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Shaofeng Yan & Xiaohong Xu a

College of Life Science, Hebei Normal University, Shijiazhuang City, Hebei Province, People’s Republic of China,

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Hebei Fitness Biotechnology Co., Ltd., Shijiazhuang High-tech Industrial Park, Shijiazhuang City, Hebei Province, People’s Republic of China, and c

College of Environmental Science and Engineering, Hebei University of Science and Technology, Shijiazhuang, China Published online: 25 Jul 2015.

To cite this article: Jintian He, Huiqi Li, Chao Liu, Gaizhen Wang, Lan Ge, Shufen Ma, Lijing Huang, Shaofeng Yan & Xiaohong Xu (2015): Formulation and evaluation of poly(lactic-co-glycolic acid) microspheres loaded with an altered collagen type II peptide for the treatment of rheumatoid arthritis, Journal of Microencapsulation: Micro and Nano Carriers To link to this article: http://dx.doi.org/10.3109/02652048.2015.1065924

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http://informahealthcare.com/mnc ISSN: 0265-2048 (print), 1464-5246 (electronic) J Microencapsul, Early Online: 1–10 ! 2015 Informa UK Ltd. DOI: 10.3109/02652048.2015.1065924

RESEARCH ARTICLE

Formulation and evaluation of poly(lactic-co-glycolic acid) microspheres loaded with an altered collagen type II peptide for the treatment of rheumatoid arthritis Jintian He1, Huiqi Li1, Chao Liu2, Gaizhen Wang3, Lan Ge2, Shufen Ma1, Lijing Huang2, Shaofeng Yan2, and Xiaohong Xu2

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College of Life Science, Hebei Normal University, Shijiazhuang City, Hebei Province, People’s Republic of China, 2Hebei Fitness Biotechnology Co., Ltd., Shijiazhuang High-tech Industrial Park, Shijiazhuang City, Hebei Province, People’s Republic of China, and 3College of Environmental Science and Engineering, Hebei University of Science and Technology, Shijiazhuang, China Abstract

Keywords

The aim of this research was to evaluate the potential of water-in-oil-in-water (w/o/w) and solid-in-oil-in-water (s/o/w) emulsification techniques to prepare the altered collagen type II peptide AP268-270 (ACTP)-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres to make ACTP more convenient as an rheumatoid arthritis treatment. Microspheres produced by the s/o/w method had higher drug encapsulation efficiency (69.7–79.8%) than those prepared by the w/o/w method (21.8–39.3%). In vitro drug release was influenced by the microencapsulation technique, molecular weight, and composition of the polymer. After intramuscular injection of the optimal formulation to Lewis rats, the concentration of ACTP peptide in serum reached its maximum level on day 3 and then remained nearly stable for approximately 4 weeks. In a collagen-induced arthritis rat model, a single intramuscular injection of ACTPloaded PLGA microspheres had comparable efficacy to the intravenous injection of ACTP peptide solution once every other day.

Altered peptide, collagen type II, PLGA microspheres, controlled release, pharmacokinetics, therapeutic studies, rheumatoid arthritis

Introduction Rheumatoid arthritis (RA) is a type of chronic autoimmune disease that causes inflammation of peripheral joints and subsequently, leads to the erosion of cartilage and bone. RA is not a life-threatening disease; however, it is a progressive illness that can lead to the destruction of joints and cause functional disability. Currently, RA is treated by using disease-modifying, anti-rheumatic drugs, and biological therapies, which have significantly improved the patient’s quality of life (Senolt et al., 2009; Yuan et al., 2012). Biological therapies targeting specific mediators of RA-induced inflammation such as tumor necrosis factor (TNF), interleukin (IL)-6, T cells, and B cells have improved RA treatment. However, the use of existing drugs does not result in long-term, drug-free remission for most patients, and lifelong treatment is generally needed. Approximately 30% of RA patients do not reach the desired clinical outcomes, and for patients for whom treatment fails, the progression of RA may result in joint deformity, disability, and loss of productivity (Emery and Dorner, 2011; Thomas, 2013). Moreover, most existing drugs are non-specific inhibitors of inflammatory

Address for correspondence: Jintian He, College of Life Science, Hebei Normal University, NO.20 Road East of 2nd Ring South, Shijiazhuang City, Hebei Province 050024, People’s Republic of China. Tel: 86-311-80787570. E-mail: [email protected] Gaizhen Wang, College of Environmental Science and Engineering, Hebei University of Science and Technology, Shijiazhuang 050018, China. Tel: 86-311-85895276. E-mail: [email protected]

History Received 21 January 2015 Revised 1 May 2015 Accepted 4 June 2015 Published online 20 July 2015

pathways or immune cells and may cause undesired side-effects, including serious infections (Keyser, 2011). Thus, there is a clear need to develop novel treatments for RA. Although the pathogenesis of RA is still not fully understood, over the past few decades, there have been significant advances in the understanding of the pathophysiology of RA. The disease has been genetically associated with the class II major histocompatibility complex (MHC-II) proteins HLA-DR1 and HLA-DR4 (Deighton et al., 1993; Luckey et al., 2014). These MHC-II molecules can present antigenic peptides to CD4+ helper T cells, which results in the initiation of autoimmune responses. It has been observed that collagen type II (CII) is one of the autoantigens associated with RA, and that CII263-272 peptide is the predominant T cell epitope in CII (Ba¨cklund et al., 2002; Qian et al., 2010). Several studies have indicated that the altered CII263-272 peptides that have consecutive or single substitutions of the T cell receptor (TCR)-contacting residues can potentially inhibit the T-cell response induced by wild-type CII peptide (Zhou et al., 2003; Cheng et al., 2005; Li et al., 2006; Kimata et al., 2012). In vivo studies have demonstrated that altered CII263-272 peptides can prevent the progression of arthritis in the collagen-induced arthritis (CIA) rat model suggesting that altered CII peptide might be a novel therapeutic agent against RA (Myers et al., 2004; Yao et al., 2007; Katsara et al., 2008; Li et al., 2009). Among the altered CII261-273 peptides, AP268-270 (ACTP) is the most potent T cell activation suppressor. In this peptide, the major T cell receptor binding amino acids 268Q, 269 P, and 270 K are replaced with alanine, glycine, and alanine, respectively

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(Cheng et al., 2005). Several studies have also demonstrated that ACTP peptide can effectively reduce joint inflammation and joint injuries in CIA rat model (Yao et al., 2006, 2007). Thus, ACTP peptide may be a promising therapeutic agent for treating RA. However, like other small peptides, ACTP peptide has a relative short serum half-life and requires long-term frequent injections for therapeutic efficacy. This need for frequent injections makes this treatment somewhat impractical. Therefore, a formulation that allows the sustained delivery of ACTP over a longer period of time is desirable. Biodegradable, biocompatible poly(D,L-lactic-co-glycolic acid) (PLGA) microparticles are one of the most promising methods of enabling long-acting peptide and protein delivery (Mundargi et al., 2008; Ye et al., 2010). Injectable depot formulations of PLGA can allow controlled drug release over several days or even months, and this significantly increases both the patients’ comfort and their compliance. Several PLGA depot products containing peptide hormones and other drugs have become commercially available, for example, Lupron DepotÕ , TrelstarÕ Depot, and RisperdalÕ ConstaÔ. Water-in-oil-in-water (w/o/w) and solid-in-oil-in-water (s/o/w) emulsion solvent evaporation techniques have been widely studied as a means of encapsulating therapeutic proteins, peptides, and vaccines (Jiang et al., 2005; Ye et al., 2010). Using the w/o/w preparation technique, the peptide is dissolved in an aqueous solution before being mixed with the polymer. Ordinarily, hydrophilic drugs can be efficiently encapsulated into PLGA microspheres by using the w/o/w method. Using the s/o/w method, the encapsulated drug is directly dispersed in the polymer phase as a solid powder. The s/o/w method can more effectively maintain the stability of the encapsulated proteins, increase the drug entrapment efficiency, and decrease burst release than the w/o/w method (Lamprecht et al., 2000; Xu et al., 2009). In this work, both the s/o/w and the w/o/w emulsion techniques were used to encapsulate ACTP peptide into PLGA microspheres. The effect of the different emulsion techniques on microparticle size, microsphere morphology, encapsulation efficiency, in vitro release, and polymer erosion kinetics was investigated and compared. The most effective ACTP-loaded PLGA microsphere formulation was determined and then used to evaluate both the in vivo pharmacokinetics in Lewis rats and the therapeutic studies of the ACTP-loaded PLGA microspheres in a CIA rat model.

Materials and methods Materials The altered CII peptide AP268-270 (ACTP) (Phe-Lys-Gly-GluGln-Ala-Gly-Ala-Gly-Glu) was obtained from Hebei Fitness Biotechnology Development Co., Ltd. (Shijiazhuang, China). Poly(vinyl alcohol) (PVA) with a molecular weight range of 31 000–50 000 Da was obtained from Aldrich Chemical Co., Inc. (Milwaukee, WI). PLGA, a copolymer with a LA/GA ratio of 50:50 or 75:25 and with an average molecular weight of 9 kDa, 13 kDa or 23 kDa) was purchased from Jinan Daigang Biomaterial Co., Ltd. (Jinan, China). Bovine CII and Freund’s incomplete adjuvant were purchased from Sigma-Aldrich Corporation (Milwaukee, WI). All the chemicals used were of analytical grade. Preparation of microspheres ACTP-loaded microspheres were fabricated using either the w/o/w or the s/o/w emulsification solvent evaporation method as previously described (He et al., 2006, 2011). To prepare the s/o primary suspension, lyophilised ACTP peptide powder with a particle size of about 1–2 mm (10 mg; ‘‘s’’) was dispersed in PLGA (2.5 mL; 120 mg/mL solution in methylene chloride; ‘‘o’’)

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by extensive stirring with a magnetic stirrer at 24 000 rpm for 1.5 min (Ultraturrax T25 basic, Ika-Werke, Staufen, Germany). The w/o primary emulsion was prepared by emulsifying an aqueous ACTP peptide solution (10 mg in 0.25 mL of 20 mM glycine-NaOH buffer, pH 9.8; ‘‘w’’) into PLGA (2.5 mL; 120 mg/ mL in methylene chloride; ‘‘o’’) by stirring with a magnetic stirrer at 24 000 rpm for 1.5 min. The w/o emulsion and the s/o primary suspension were added to a 0.5% (w/v) aqueous PVA solution (150 mL) containing 5.0% NaCl and then homogenised at 600 rpm for 1 min. For solvent extraction, the w/o/w and the s/o/w emulsions were subsequently stirred with a magnetic stirrer at 300 rpm for 6 h. The resulting ACTP-containing microspheres were collected by filtration and washed three times with distilled water. The microspheres were then vacuum-dried over night and stored at 20  C until use. Particle size and morphology of the ACTP-loaded PLGA microspheres The morphology and size of the PLGA microspheres were examined by using scanning electron microscopy (SEM, Hitachi S-520, Hitachi Ltd., Tokyo, Japan). The PLGA microspheres were mounted onto metal stubs by using double-sided adhesive tape and vacuum-coated with a thin layer of gold. The samples were then examined by using SEM. The diameter of 100, randomly selected PLGA microspheres was measured, and the results were presented as an average. In vitro evaluation of ACTP peptide release and polymer degradation behaviour of the ACTP-loaded PLGA microspheres Dried microspheres (30 mg) were suspended in phosphatebuffered saline (PBS; 1 mL; pH 7.4) containing 0.05% (w/v) sodium azide and placed in a SKY-211D rocking incubator (Zhicheng, China) operating at 150 rpm at 37  C. At each sampling time, the supernatant was withdrawn and replaced with the same volume of fresh PBS solution. The ACTP peptide concentration in the supernatant was assayed by using the HPLC method described in this section. The amount of ACTP peptide released within 24 h was defined as the initial burst. At predetermined time intervals, microspheres were centrifuged at 5000 rpm (Eppendorf MiniSpin plus, Eppendorf AG, Hamburg, Germany), washed with distilled water, and subsequently lyophilised. The surface morphology of these microspheres was examined using SEM. Mass loss of the microspheres was evaluated gravimetrically. Pharmacokinetics of the ACTP-loaded PLGA microspheres in Lewis rats The in vivo release of the ACTP-loaded PLGA microspheres was evaluated in female Lewis rats (female, 200–220 g) (Vital River Laboratories, Beijing, China). The rats were housed under conventional laboratory conditions in a room maintained at 24 ± 1  C and fed commercial rat food and water ad libitum. All animal studies were performed at Hebei Normal University in accordance with Institutional Animal Care and Use Committee guidelines. The rats were randomly divided into three groups of six rats each. One group was used for intravenous injection of ACTP peptide solution at a dose of 3.0 mg/kg, the second and the third groups were used for intramuscular injection of the microspheres at a dose of 30 or 15 mg/kg of ACTP peptide. The ACTP-loaded microspheres were pre-suspended in a diluent containing 2% carboxymethylcellulose sodium and 0.9% (w/v) NaCl. After injection, 0.4 mL of blood was withdrawn from the retro-orbital sinus through the eye canthus of anesthetised rats at

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0.083, 0.25, 0.75, 2, 3, 5, 7, 9, and 24 h after injection of the ACTP peptide solution and at 1, 2, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, and 30 d after injection of the microsphere formulations. The blood samples were collected in a pyrogen-free heparincontaining tube and centrifuged at 1500 rpm for 15 min. Approximately 0.2 mL of plasma samples was obtained, which was collected in a silicone-coated tube and stored at 40  C. The concentrations of ACTP peptide in the plasma samples were determined by using HPLC (section ‘‘Therapeutic studies of the ACTP-loaded . . .’’). The pharmacokinetic analysis of the data was performed by using the Practical Pharmacokinetic Program 3P87 specially developed by the Chinese Pharmacological Society. The following pharmacokinetic parameters were determined: the area under the curve from time zero to last sampling time (AUC0–t), mean residence time (MRT), and the apparent elimination rate constant (Kel). High-performance liquid chromatography (HPLC) assays for determination of peptide concentration The ACTP peptide content of the microspheres was determined by using an extraction method (He et al., 2011). Dried microspheres (30 mg) were dissolved in methylene chloride. After centrifugation at 10 000 rpm for 15 min and removal of the polymer solution, the remaining pellet was dissolved in 20 mM phosphate buffer (0.2 mL; pH 7.4). After centrifugation at 10 000 rpm for 15 min, the concentrations of ACTP peptide in the supernatant were determined by using the HPLC method described below. HPLC analyses were performed with a Shimadzu LC 10Avp system (Shimadzu, Kyoto, Japan) by using a Dikma Diamonsil C18 column (250 mm  4.6 mm, 5 mm). The mobile phase consisted of 40% (v/v) acetonitrile containing 0.1% trifluoroacetic acid in water and the flow rate was 1.0 mL/min. The absorbance was recorded at 215 nm. ACTP peptide showed a retention time of approximately 12.25 min. The amount of ACTP peptide was taken into account in the calculation of the actual peptide loading (mg of encapsulated peptide per 100 mg of microspheres). The encapsulation efficiency of ACTP peptide in the microspheres was calculated as the ratio of actual and theoretical peptide loadings. The concentration of ACTP peptide in plasma was also determined with HPLC with a slight modification. A Venusil XBP ˚ ; Bonna-Agela C18 column (4.6 mm  250 mm, 5 mm, 300 A Technologies, Tianjin, China) was employed. ACTP peptide showed a retention time of approximately 17.5 min. A calibration curve was constructed by using blank rat plasma with nine known concentrations of ACTP peptide. The linear concentration range of the calibration curve was 0.30–60.0 mg/mL of ACTP peptide. Concentrations of 460.0 mg/mL were determined by diluting samples into the linear range. The regression equation of the calibration curve was y ¼ 2.7947x, r2 ¼ 0.998. The limit of quantification (LOQ) was 0.125 mg/mL. Therapeutic studies of the ACTP-loaded PLGA microspheres in rats The therapeutic studies of the ACTP-loaded PLGA microspheres were evaluated in CIA model of rats. Female Lewis rats (6- to 7-weeks-old, 175–200 g) were used, and CIA was induced as described previously (Waksman et al., 1996). Briefly, Bovine CII was dissolved in 0.1 M acetic acid at a concentration of 4 mg/mL and emulsified with Freund’s incomplete adjuvant. On day 0, each rat was injected intradermally at the base of the tail with 75 mL of the emulsion containing 150 mg of CII. On day 7, a second

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injection of CII emulsion was administered at the base of the tail. The onset of arthritis in the ankle joints could usually be recognised visually between days 12 and 14. The CIA rats were randomly divided into four groups of six rats each. The treatment with ACTP peptide was initiated 24 h after the onset of CIA (Yao et al., 2006, 2007). One group was subjected to intravenous injection of ACTP peptide solution at a dose of 1.0 mg/kg once every other day for 3 weeks, the second group was subjected to intramuscular injection of the microspheres at a dose of 15.0 mg/kg of ACTP peptide, the third group was subjected to intramuscular injection of the equivalent amount of blank microspheres (without ACTP peptide), and the fourth group was subjected to intravenous injection of the equivalent amount of PBS solution as a control. The incidence and severity of CIA were determined by visually inspecting each paw and scoring based on the degree of swelling, erythema, and deformation of the joints (Kim et al., 2004). Arthritic lesions on each paw were scored on a scale of 0–4: 0 ¼ no change, 1 ¼ swelling and erythema of the digit, 2 ¼ mild swelling and erythema of the limb, 3 ¼ gross swelling and erythema of the limb, 4 ¼ gross deformity and inability to use the limb. The cumulative score for each rat was calculated by adding the scores for the individual paws. The maximum possible score for each rat was 16. Statistical analysis The differences between two groups were evaluated statistically by using analysis of variance (ANOVA) followed by the Newman–Keuls test. A p value of 50.05 was considered to be statistically significant. All analysis of the data was performed by using the statistical software package (version 6.0, StatSoft Inc., Tulsa, OK).

Results and discussion Characteristics of the ACTP-loaded PLGA microspheres Morphological examination of the PLGA microspheres by using SEM showed that both encapsulation techniques produced smooth and spherical surfaces without visible pores at the surface (Figure 1, day 0). An investigation of the internal structure indicated that the microspheres prepared by using the s/o/w method had a compact interior while the w/o/w microspheres had a loose and porous interior (Figure 2). The drug encapsulation efficiency of the ACTP-loaded PLGA microspheres produced by using the w/o/w method was generally low and ranged from 22.0% to 39.3% (Table 1). The presence of NaCl in the external water phase significantly affected the properties of the ACTP-loaded microspheres. When the concentration of NaCl in the external water phase was increased from 0 to 5%, the drug encapsulation efficiency of the microspheres made with 9 kDa PLGA (50:50) increased from 22.0% to 35.0%, while the initial burst release decreased from 80.4% to 12.2%. It has been suggested that salt in the external water phase affected the drug encapsulation efficiency and the initial burst release by balancing the osmotic pressure between the internal and external water phases. In the w/o/w method prepared ACTP-loaded microspheres, the osmotic pressure led to an influx of water from the external water phase and resulted in porous, larger microspheres (Figure 3, A1 and Table 1). The peptide could rapidly escape through these pores and this resulted in a high initial burst release (Table 1). However, when NaCl was added to the external water phase, the osmotic pressure was balanced, giving rise to denser, smaller microspheres. The large pores at the surface of microspheres disappeared and the initial burst release was also significantly reduced (Figure 3, A2 and Table 1). To further improve the drug encapsulation efficiency, 13 kDa (75:25)

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(B)

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(C)

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Figure 1. Scanning electron microscopic images of the ACTP-loaded poly(lactic-co-glycolic acid), PLGA microspheres during incubation in phosphate buffered saline at 37  C. Note: 9 kDa PLGA (A), 13 kDa PLGA (B), and 23 kDa PLGA (C) microspheres prepared by using the water-in-oil-in-water or solid-in-oil-in-water methods corresponding to formulation B, D-I in Table 1, respectively.

or 23 kDa PLGA (75:25) was used to fabricate ACTP-loaded microspheres. However, the drug encapsulation efficiency did not increase significantly (Table 1). PLGA microspheres prepared using the s/o/w method achieved significantly higher drug encapsulation efficiency (69.7–79.8%) than those made with the w/o/w method (22.0–39.3%) (Table 1). The difference observed in the peptide encapsulation efficiency

between the different preparation methods could be explained by the leakage of the encapsulated peptide into the external aqueous phase during the encapsulation process (Lamprecht et al., 2000). In the w/o/w method, shear forces were used to disperse the w/o primary emulsion into the external aqueous phase to form a w/o/w double emulsion. First, the splitting of w/o primary emulsion led to an immediate loss of drug. Second, to increase the solubility of

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Figure 2. Scanning electron microscopic images of the internal morphology of the ACTP-loaded poly(lactic-co-glycolic acid), PLGA microspheres. Note: A1 and A2: PLGA microspheres prepared by using the water-in-oil-in-water method corresponding to formulation B and E in Table 1, respectively; B1 and B2: microspheres prepared by using the solid-in-oil-in-water method corresponding to formulation D and F in Table 1, respectively.

A1

A2

B1

B2

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Table 1. Characterisation of the ACTP-loaded poly(lactic-co-glycolic acid) microspheres prepared by water-in-oil-water double emulsion solvent evaporation method (n ¼ 3). Batch A B C D E F G H I

Method

MW of PLGA

Ratio of LA:GA

NaCl (%)

Particle size (mm)

DL (%)

EE (%)

IB (%)

w/o/w w/o/w s/o/w s/o/w w/o/w s/o/w s/o/w w/o/w s/o/w

9K 9K 9K 9K 13K 13K 13K 23K 23K

50:50 50:50 50:50 50:50 75:25 75:25 50:50 75:25 75:25

– 5 – 5 5 5 5 5 5

87.3 ± 4.5 68.4 ± 4.2 55.9 ± 3.8 59.3 ± 3.1 76.0 ± 5.1 73.5 ± 34.2 63.5 ± 3.9 86.8 ± 5.2 78.7 ± 4.6

0.71 ± 0.03 1.13 ± 0.06 2.40 ± 0.12 2.38 ± 0.09 1.24 ± 0.08 2.58 ± 0.10 2.25 ± 0.09 1.27 ± 0.12 2.38 ± 0.11

22.0 ± 1.2 35.0 ± 1.9 74.3 ± 3.2 73.7 ± 3.6 38.4 ± 2.9 79.8 ± 4.2 69.7 ± 3.9 39.3 ± 1.9 73.7 ± 4.6

80.4 ± 4.5 12.2 ± 1.3 18.2 ± 1.3 13.5 ± 0.9 19.6 ± 1.2 15.2 ± 0.8 14.7 ± 0.9 12.4 ± 0.7 13.5 ± 0.5

Notes: MW, Weight average molecular weight; DL, drug loading; LA, lactide; GA, glycolide; EE, encapsulation efficiency; IB, initial burst.

ACTP peptide, the pH of the internal water phase was set at 9.8, at which a PLGA molecule would carry a negative net charge. The electrostatic repulsion between PLGA units would result in unstable w/o microemulsions within the w/o/w double emulsions and therefore, enhance the loss of the drug. At the same time, the instability of w/o microemulsions would lead to coalescence of w/o microemulsions and the formation of large pores within the microspheres (Figure 2A). In case of the s/o/w method, the ACTP peptide was in the solid state and required a dissolution step before it could leak into the external aqueous phase. Therefore, the ACTP peptide release from the organic phase should be slower than for the w/o/w method. On the other hand, the external aqueous phase (pH 6.0) was slightly acidic and was close to the isoelectric point (pI: 4.9) of ACTP peptide. The solubility of ACTP peptide at this pH value (near pI) is low. Thus, peptide leakage from s/o suspension was greatly reduced, and the peptide encapsulation efficiency of the microspheres prepared using the s/o/w method was significantly increased. As shown in Table 1, the initial burst of the s/o/w microspheres significantly decreased when 5% NaCl was added to the external water phase. The initial burst release was mainly attributed to the release of peptide adsorbed on the surface and diffused from the

pores on the surface of microspheres (Huang and Brazel, 2001). With NaCl added into the outer water phase, the material exchange between the inner and outer phases was inhibited (Zhou et al., 2010; He et al., 2011). The pores on the surface of microspheres disappeared, and the initial burst release through the pores on the surface of microparticles subsequently decreased (Figure 3B and Table 1). In vitro release of the ACTP peptide and degradation of PLGA microspheres The in vitro cumulative release profiles of ACTP peptide from the ACTP-loaded PLGA microspheres are shown in Figure 4(A). The preparation method, molecular weight of the polymer, and the lactide/glycolide ratio in the PLGA significantly affect the release behaviour. The release profile of microspheres made with 9 kDa PLGA (50:50) showed that more than 95% of ACTP peptide was continuously released from the microspheres over 20 d, independent of the microencapsulation technique used (Figure 4A). A plot of ACTP peptide release from the 9 kDa PLGA (50:50) microspheres prepared by using the w/o/w method almost

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Figure 3. Scanning electron microscopic images of the ACTP-loaded poly(lacticco-glycolic acid), PLGA microspheres prepared with 0% (A1, B1) and 5.0% (A2, B2) NaCl in the external phase of solid-in-oil-inwater (s/o/w) or water-in-oil-in-water (w/o/w) emulsion. Note: A1 and A2: PLGA microspheres prepared by using the w/o/w method corresponding to formulation A and B in Table 1, respectively; B1 and B2: microspheres prepared using the s/o/w method corresponding to formulation C and D in Table 1, respectively.

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Figure 4. In vitro release of the ACTP-loaded poly(lactic-co-glycolic acid), PLGA microspheres in phosphate-buffered saline at 37  C. Note: (A) Comparing the release behaviour of different PLGA microspheres. (B) The cumulative release of ACTP peptide from PLGA microspheres as a function of the square root of time.

corresponds to zero-order kinetics. The peptide release rate from the microspheres prepared by using the s/o/w method was clearly faster than those prepared by using the w/o/w method, especially during the early stages of release. A plot of cumulative release of peptide versus the square root of time demonstrated a linear relationship over 12 d for the microspheres prepared with the s/o/ w method (Figure 4B); approximately 84% of the ACTP peptide was released during this period. Thus, the mechanism responsible for the release of ACTP peptide release from 9 kDa PLGA (50:50) microspheres prepared using the s/o/w method is mainly diffusion through the aqueous channels/pores (Peppas, 1985). To further investigate the release process, particle degradation was investigated by using SEM and by assessing the mass loss of particles. The results are shown in Figures 1 and 5. The erosion rate of 9 kDa PLGA (50/50) microspheres prepared by using the w/o/w method was slightly faster than those prepared by using the s/o/w method (Figure 5). However, the microspheres prepared with the s/o/w method developed many pores on the surface early on the fifth day while the microspheres prepared by the w/o/w method generated large pores later on the 10th day (Figure 1A). These results were consistent with the faster release rate of peptide from microspheres prepared with the s/o/w method early in the release process. The structure of the PLGA microspheres was heavily damaged after 20 d, but some residual structure could still be observed (Figure 1A). The results suggested that ACTP release from 9 kDa PLGA (50:50) microspheres prepared by the w/o/w method occurs by a combination of diffusion through the aqueous channels/pores and degradation of the polymer matrix (Fredenberg et al., 2011). For the 13 kDa PLGA (75/25) and 13 kDa PLGA (50/50) microspheres, the microencapsulation method greatly influenced the ACTP peptide release profiles. More than 95% of the total ACTP peptide was released within 30 d from the 13 kDa PLGA (75/25) microspheres prepared by the w/o/w method (Figure 4A). The release profile showed a triphasic release pattern, which is typical of PLGA microspheres; this involves an initial burst release, followed by a lag phase, and then a secondary burst

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Figure 5. Weight loss of the ACTP-loaded microspheres incubated in phosphate-buffered saline at 37  C. Note: PLGA microspheres prepared by using the water-in-oil-water or solid-in-oil-in-water methods corresponding to formulation B, D-I in Table 1, respectively.

release phase. After the initial burst, the peptide in the interior of microspheres might slowly diffuse from the pores at the surface resulting in the lag phase observed in the release profile (Fredenberg et al., 2011). The lag phase continued until the polymer was degraded into water-soluble oligomers (Shah et al., 1992). After that, the ACTP peptide was released more rapidly from the interior of the microspheres. As shown in the SEM images, many pores developed on the surface of the 13 kDa PLGA (75/25) microspheres prepared using the w/o/w method at day 10, which corresponded to the beginning of the second burst release (Figure 1B). On the contrary, the 13 kDa PLGA (75/25) and 13 kDa PLGA (50/50) microspheres prepared by the s/o/w method did not display an obvious lag phase after initial burst release. As can be seen in the SEM images in Figure 1(B), the 13 kDa PLGA (50/50) and 13 kDa PLGA (75/25) microspheres prepared by the s/o/w method developed many pores on the surface around day 5. ACTP peptide could have easily escaped from these pores suggesting that the release did not slow down after the initial burst. The release profiles of 13 kDa PLGA (50/50) microspheres prepared by the s/o/w method had a biphasic release profile: (1) a rapid release of about 38% of the total ACTP occurred within the first 3 d; (2) after that, about 52% of the total peptide was released in a sustained manner according to near zero-order kinetics for 27 d (Figure 4A). The dry weight decreased to 46% of the initial value of the microspheres (Figure 5). These results suggested that ACTP peptide release from 13 kDa PLGA (50/50) microspheres might be controlled by a combination of diffusion through the aqueous pores and erosion of the polymer matrix (Fredenberg et al., 2011). As shown in Figure 4(A), the release profiles of ACTP peptide from the 23 kDa PLGA (75/25) microspheres were similar to that of the 13 kDa PLGA (75/25) microspheres prepared with the w/o/w method. The SEM images shown in Figure 1(C) indicated that no obvious pores appeared on the surface of the 23 kDa PLGA (75/25) microspheres prepared by the s/o/w method or w/o/w method until the 20th day. Thereafter, numerous pores formed on the surface of the 23 kDa PLGA (75/25) microspheres (Figure 1C), which corresponded to the accelerated release of peptide during the secondary burst release phase (Figure 4A). Based on the above data, the 13 kDa PLGA (50/50) microspheres prepared with the s/o/w method, and possessed high encapsulation efficiency and a near zero-order drug release

Figure 6. (A) The ACTP peptide plasma levels after intravenous injection of ACTP peptide solution at a dose of 3.0 mg ACTP peptide/kg body weight in Lewis rats. (B) The ACTP peptide plasma levels after intramuscular injection of poly(lactic-co-glycolic acid), PLGA microspheres in Lewis rats: 30 mg ACTP peptide/kg body weight (g) and 15 mg ACTP peptide/kg body weight (m). Note: The ACTP-loaded PLGA microspheres corresponding to formulation G in Table 1. Mean ± standard deviation (SD). n ¼ 6.

profile for 27 d, were selected for further pharmacokinetic and therapeutic studies. In vivo pharmacokinetic studies Figure 6(A) shows the in vivo concentration–time profiles of ACTP after intravenous injection of a solution of ACTP peptide to rats. The rapid decrease in ACTP peptide concentration indicated that rapid elimination of ACTP peptide took place. The rapid decrease in the concentration of ACTP peptide suggested that frequent injections would be needed to maintain therapeutic levels of ACTP peptide in plasma. The ACTP peptide plasma concentration–time data were fit by using a one-compartment model with the 3p87 computer program. As shown in Table 2, the MRT and the Kel after intravenous injection were determined to be 0.16 d and 6.36 d1, respectively. Figure 6(B) shows the ACTP peptide plasma concentration– time profile after intramuscular injection of the ACTP-loaded PLGA microspheres. The ACTP peptide plasma concentration increased continuously during the initial 3 d. The peak plasma concentration (Cmax) was 3014 ng/mL and 1317 ng/mL for the microsphere formulation at a dose of 30 mg ACTP peptide/kg body weight and 15 mg ACTP peptide/kg body weight, respectively. Three days later, the ACTP peptide concentrations decreased gradually and remained nearly constant from day

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Table 2. Pharmacokinetic parameters of ACTP solution and the ACTP-loaded microspheres (MS) after intravenous or intramuscular injection to female Lewis rats, respectively (n ¼ 6). Samples Solution containing 3 mg ACTP/kg body weight MS containing 30 mg ACTP/kg body weight MS containing 15 mg ACTP/kg body weight

Kel (d1)

MRT(d)

AUC0–t (ngd/ml)a

Bioavailability (%)

6.36 ± 0.12 0.56 ± 0.08 0.51 ± 0.09

0.160 ± 0.02 14.7 ± 1.8 13.9 ± 2.1

4227.1 ± 313.2 36098.4 ± 610.5 18471.3 ± 2546.8

100 85.5 87.4

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Notes: Mean ± standard deviation (SD). A one-compartment model was used to calculate the pharmacokinetic parameters of ACTP peptide solution and a non-compartment pharmacokinetic method was employed to determine the pharmacokinetic parameters of the ACTP-loaded poly(lactic-co-glycolic acid) microspheres. a The area under curve (AUC) was calculated from 0 to 24 h for the ACTP peptide solution and from 0 to 30 d for the ACTP-loaded PLGA microspheres.

Figure 7. Correlation of in vitro and in vivo release of ACTP peptide from the ACTP-loaded poly(lactic-co-glycolic acid), PLGA microsphere.

7 to day 25. Thereafter, the peptide plasma concentration increased again at day 27 and then declined rapidly at day 30. The ACTP peptide plasma concentration was sustained for 30 d, which was similar to the in vitro release data. After analysing the concentration–time profiles, the pharmacokinetic parameters were determined by using a non-compartment model and are summarised in Table 2. The apparent Kel of ACTP peptide release from PLGA microspheres was estimated to be 0.56 day1 and 0.51 day1 for the microsphere formulation administered at a dose of 30 mg ACTP peptide/kg and 15 mg ACTP peptide/kg, respectively (Table 2). When compared to the ACTP peptide solution, the Kel value decreased by a factor of 11.4 and 12.5 for the microspheres administered at 30 mg ACTP peptide/kg and 15 mg ACTP peptide/kg, respectively. The MRT was determined to be 14.7 d and 13.9 d for the microsphere formulation administered at a dose of 30 mg ACTP peptide/kg and 15 mg ACTP peptide/kg, respectively (Table 2). The AUC0–t were 36 098.4 and 18 471.3 ngd/mL for the microsphere formulations administered at a dose of 30 mg ACTP peptide/kg and 15 mg ACTP peptide/kg, respectively. The bioavailability of ACTP peptide released from PLGA microspheres administered at a dose of 30 mg ACTP peptide/kg and 15 mg ACTP peptide/kg was 85.5% and 87.4%, respectively. It is important to consider the in vitro and in vivo correlation (IVIVC) when developing parenteral biodegradable depot systems (Chu et al., 2006; Sun et al., 2008). A proper IVIVC can be used to predict the in vivo release profile of parenteral biodegradable depot systems based on an in vitro release profile conducted in PBS. The data obtained from the in vivo release of the microspheres administered at a dose of 15 mg ACTP peptide/kg was used to develop an IVIVC and the result is shown in Figure 7.

Figure 8. Inhibition of arthritis in Lewis rats with collagen-induced arthritis by treatment with different ACTP peptide formulation: intravenous injection of ACTP peptide solution at a dose of 1.0 mg/kg body weight once every other day (m); intramuscular injection of the microspheres at a dose of 15.0 mg ACTP/kg body weight within the poly(lactic-co-glycolic acid), PLGA microspheres () and the equivalent amount of blank microspheres (without ACTP) as a control (g). Note: The ACTP-loaded PLGA microspheres corresponding to formulation G in Table 1. Mean ± standard deviation (SD). n ¼ 6.

A good linear regression relationship was obtained between the percentage of ACTP peptide released in vitro in PBS at 37  C and the percentage AUC after intramuscular administrations of the ACTP-loaded microspheres to rats (R2 ¼ 0.98). However, the percentage AUC (2.4%) was significantly lower than the percentage released in vitro (15.3%) after the initial burst phase. A similar phenomenon has also been observed in the IVIVC of ethinyl estradiol- and gestodene-loaded PLGA microspheres (Sun et al., 2008). The complexity of the in vivo environment might influence the release of drugs from the PLGA microspheres in the intramuscular site during the burst phase. After the initial burst, the release behaviour of PLGA microspheres administrated intramuscularly was similar to the in vitro release. This is consistent with the observation that degradation of PLGA microspheres administrated intramuscularly is mainly due to the hydrolytic degradation, which is similar to the in vitro situation (Sandor et al., 2002). The in vivo therapeutic studies The in vivo therapeutic studies of the ACTP-loaded PLGA microspheres were examined by measuring the mean arthritis severity score in CIA rats and the results are illustrated in Figure 8. The CIA was progressive and the mean arthritis severity score continuously increased from 2.2 on the first day after

DOI: 10.3109/02652048.2015.1065924

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arthritis onset to 7.8 at 21 day for PBS group. To confirm that blank PLGA microspheres has no effect on the progress of CIA, PLGA microspheres without ACTP peptide were administered, and the mean arthritis severity score was measured. No significant differences in mean arthritis severity scores were observed between the PBS and the blank PLGA microsphere groups (p40.05) indicating that blank PLGA microspheres had no effect on the progress of CIA (Figure 8). The continuous injection of ACTP peptide solution clearly inhibited the development of CIA, and the increase of the corresponding mean arthritis scores was significantly slower than that of the PBS group, after treatment with ACTP peptide solution for 3 d (p50.05). At the same time, a single injection of ACTP-loaded microspheres significantly decreased the mean arthritis severity scores equivalent to the continuous injection of ACTP peptide solution (p50.05). These results suggested that the ACTP-loaded microsphere formulation clearly inhibits the progress of CIA.

Conclusion This research evaluated the potential of the w/o/w and s/o/w methods to prepare ACTP-loaded microspheres for the treatment of RA. Use of the s/o/w method allowed a significantly higher degree of drug encapsulation efficiency than the w/o/w method. The preparation method, molecular weight of the polymer, and the lactide/glycolide ratio of PLGA significantly affected the drug release and polymer degradation behaviour of the ACTP-loaded PLGA microspheres. The pharmacokinetics of the ACTP-loaded microspheres in Lewis rats indicated that serum ACTP peptide concentration could be roughly maintained at steady levels for approximately 4 weeks. A good linear correlation (R2 ¼ 0.98) was obtained between the in vitro and in vivo release data. A single intramuscular injection of the ACTP-loaded PLGA microspheres to rats resulted in an effective reduction of the joint inflammation and a decrease of the joint injuries in CIA. These results suggested that the ACTP-loaded PLGA microspheres might be potentially useful in the treatment of RA.

Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the article.

References Ba¨cklund J, Carlsen S, Ho¨ger T, Holm B, Fugger L, Kihlberg J, Burkhardt H, Holmdahl R. Predominant selection of T cells specific for the glycosylated collagen type II epitope (263–270) in humanised transgenic mice and in rheumatoid arthritis. Proc Natl Acad Sci USA, 2002; 99(15):9960–5. Cheng YJ, Zhou Q, Li ZG. The inhibitory effect of altered collagen II peptide on HLA-DRB1-restricted T-cell activation. Scand J Immunol, 2005;61(3):260–5. Chu DF, Fu XQ, Liu WH, Liu K, Li YX. Pharmacokinetics and in vitro and in vivo correlation of huperzine A loaded poly(lactic-co-glycolic acid) microspheres in dogs. Int J Pharm, 2006;325(1–2):116–23. Deighton CM, Kelly PJ, Walker DJ. Linkage of rheumatoid arthritis with HLA. Ann Rheum Dis, 1993;52(9):638–42. Emery P, Dorner T. Optimising treatment in rheumatoid arthritis: A review of potential biological markers of response. Ann Rheum Dis, 2011;70(12):2063–70. Fredenberg S, Wahlgren M, Reslow M, Axelsson A. The mechanisms of drug release in poly(lactic-co-glycolic acid)-based drug delivery systems – A review. Int J Pharm, 2011;415(1–2):34–52. He J, Feng M, Zhou X, Ma S, Jiang Y, Wang Y, Zhang H. Stabilization and encapsulation of recombinant human erythropoietin into PLGA microspheres using human serum albumin as a stabilizer. Int J Pharm, 2011;416(1):69–76.

PLGA microspheres for the treatment of RA

9

He JT, Su HB, Li GP, Tao XM, Mo W, Song HY. Stabilization and encapsulation of a staphylokinase variant (K35R) into poly(lacticco-glycolic acid) microspheres. Int J Pharm, 2006;309(1–2):101–8. Huang X, Brazel CS. On the importance and mechanisms of burst release in matrix-controlled drug delivery systems. J Contr Release, 2001; 73(2–3):121–36. Jiang WL, Gupta RK, Deshpande MC, Schwendeman SP. Biodegradable poly(lactic-co-glycolic acid) microparticles for injectable delivery of vaccine antigens. Adv Drug Deliv Rev, 2005;57(2005):391–410. Katsara M, Minigo G, Plebanski M, Apostolopoulos V. The good, the bad and the ugly: How altered peptide ligands modulate immunity. Expert Opin Biol Ther, 2008;8(12):1873–84. Keyser FD. Choice of biologic therapy for patients with rheumatoid arthritis: The infection perspective. Curr Rheumatol Rev, 2011; 7(1):77–87. Kim KS, Choi YH, Kim KH, Lee YC, Kim CH, Moon SH, Kang SG, Park YG. Protective and anti-arthritic effects of deer antler aqua-acupuncture (DAA), inhibiting dihydroorotate dehydrogenase, on phosphate ions mediated chondrocyte apoptosis and rat collagen-induced arthritis. Int Immunopharm, 2004;4(7):963–73. Kimata M, Cullins DL, Brown ML, Brand DD, Rosloniec EF, Myers LK, Stuart JM, Kang AH. Characterization of inhibitory T cells induced by an analog of type II collagen in an HLA-DR1 humanized mouse model of autoimmune arthritis. Arthritis Res Ther, 2012;14(3):R107. Lamprecht A, Torres HR, Schafer U, Lehr CM. Biodegradable microparticles as a two-drug controlled release formulation: A potential treatment of inflammatory bowel disease. J Control Release, 2000;69(3):445–54. Li R, Li X, Li ZG. Altered collagen II peptides inhibited T cell activation in rheumatoid arthritis. Clin Immunol, 2006;118(2–3):317–23. Li R, Li X, Li Z. Altered collagen II 263–272 peptide immunization induces inhibition of collagen-induced arthritis through a shift toward Th2-type response. Tissue Antigens, 2009;73(4):341–7. Luckey D, Behrens M, Smart M, Luthra H, David CS, Taneja V. DRB1*0402 may influence arthritis by promoting naive CD4+ T-cell differentiation into regulatory T cells. Eur J Immunol, 2014; 44(11):3429–38. Mundargi RC, Babu VR, Rangaswamy V, Patel P, Aminabhavi TM. Nano/micro technologies for delivering macromolecular therapeutics using poly(D,L-lactide-co-glycolide) and its derivatives. J Contr Release, 2008;125(3):193–209. Myers LK, Sakurai Y, Rosloniec EF, Stuart JM, Kang AH. An analog peptide that suppresses collagen-induced arthritis. Am J Med Sci, 2004;327(4):212–16. Peppas NA. Analysis of Fickian and non-Fickian drug release from polymers. Pharm Acta Helv, 1985;60(4):110–11. Qian Z, Latham KA, Whittington KB, Miller DC, Brand DD, Rosloniec EF. An autoantigen-specific, highly restricted T cell repertoire infiltrates the arthritic joints of mice in an HLA-DR1 humanized mouse model of autoimmune arthritis. J Immunol, 2010;185(1):110–18. Sandor M, Harris J, Mathiowitz E. A novel polyethylene depot device for the study of PLGA and P(FASA) microspheres in vitro and in vivo. Biomaterials, 2002;23(22):4413–23. Senolt L, Vencovsky´ J, Pavelka K, Ospelt C, Gay S. Prospective new biological therapies for rheumatoid arthritis. Autoimmun Rev, 2009; 9(2):102–7. Shah SS, Cha Y, Pitt CG. Poly(glycolic acid-co-DL-lactic acid): Diffusion or degradation controlled drug delivery. J Contr Release, 1992; 18(3):261–70. Sun Y, Wang J, Zhang X, Zhang Z, Zheng Y, Chen D, Zhang Q. Synchronic release of two hormonal contraceptives for about one month from the PLGA microspheres: In vitro and in vivo studies. J Contr Release, 2008;129(3):192–9. Thomas R. Dendritic cells and the promise of antigen-specific therapy in rheumatoid arthritis. Arthritis Res Ther, 2013;15(1):204. doi: 10.1186/ ar4130. Waksman Y, Hod I, Friedman A. Therapeutic effects of estradiol benzoate on development of collagen-induced arthritis (CIA) in the Lewis rat are mediated via suppression of the humoral response against denatured collagen type II (CII). Clin Exp Immunol, 1996;103(3):376–83. Xu Q, Crossley A, Czernuszka J. Preparation and characterization of negatively charged poly(lactic-co-glycolic acid) microspheres. J Pharm Sci, 2009;98(7):2377–89. Yao ZQ, Li R, Li ZG. A triple altered collagen II peptide with consecutive substitutions of TCR contacting residues inhibits collagen-induced arthritis. Ann Rheum Dis, 2007;66(3):423–4.

10

J. He et al.

Downloaded by [Stockholm University Library] at 11:23 25 August 2015

Yao ZQ, Zhao JX, Li R, Li ZG. Inhibition of cartilage destruction in collagen-induced arthritis by altered CII 263-272 peptide: Experiment with rats. Zhonghua Yi Xue Za Zhi, 2006;86(43):3055–8. Ye M, Kim S, Park K. Issues in long-term protein delivery using biodegradable microparticles. J Contr Release, 2010;146(2):241–60. Yuan F, Quan LD, Cui L, Goldring SR, Wang D. Development of macromolecular prodrug for rheumatoid arthritis. Adv Drug Deliv Rev, 2012;64(12):1205–19.

J Microencapsul, Early Online: 1–10

Zhou Q, Cheng YJ, Lu HS, Zhou WH, Li ZG. Inhibition of T-cell activation with HLADR1/DR4 restricted non-T-cell stimulation peptides. Hum Immunol, 2003;64(9):857–65. Zhou XL, He JT, Zhou ZT, Ma SF, Jiang Y, Wang Y. Effect of NaCl in outer water phase on the characteristics of BSA-loaded PLGA sustained-release microspheres fabricated by a solid-in-oilin-water emulsion technique. Yao Xue Xue Bao, 2010; 45(8):1057–63.

Formulation and evaluation of poly(lactic-co-glycolic acid) microspheres loaded with an altered collagen type II peptide for the treatment of rheumatoid arthritis.

The aim of this research was to evaluate the potential of water-in-oil-in-water (w/o/w) and solid-in-oil-in-water (s/o/w) emulsification techniques to...
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