International Journal of Biological Macromolecules 69 (2014) 100–107
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Docetaxel load biodegradable porous microspheres for the treatment of colorectal peritoneal carcinomatosis RangRang Fan a,1 , YueLong Wang a,1 , Bo Han b , YouFu Luo a,∗ , LiangXue Zhou a , XiRui Peng a , Min Wu a , Yu Zheng a , Gang Guo a,∗ a State Key Laboratory of Biotherapy and Cancer Center, and Department of Neurosurgery, West China Hospital, West China Medical School, Sichuan University, Chengdu 610041, PR China b Key Laboratory of Xinjiang Phytomedicine Resources, Shihezi 832002, PR China
a r t i c l e
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Article history: Received 16 March 2014 Received in revised form 20 April 2014 Accepted 7 May 2014 Available online 20 May 2014 Keywords: PLLA–L121–PLLA Porous microspheres Colorectal peritoneal carcinomatosis
a b s t r a c t Micro- and nanoparticle formulations are widely used to improve the bioavailability of low solubility drugs. In this study, biodegradable poly(L-lactide acid)–Pluronic L121–poly(L-lactide acid) (PLLA–L121–PLLA) was developed. And then a controlled drug delivery system (CDDS), docetaxel (DOC) loaded PLLA–L121–PLLA porous microsphere (DOC MS) was prepared for colorectal peritoneal carcinomatosis (CRPC) therapy. DOC MS was prepared by DOC and PLLA–L121–PLLA using an oil-in-water emulsion solvent evaporation method. The particle size, morphological characteristics, encapsulation efficiency, in vitro drug release studies and in vitro cytotoxicity of DOC MS have been investigated. In vitro release profile demonstrated a significant difference between rapid release of free DOC and much slower and sustained release of DOC MS. Furthermore, cytotoxicity assay indicated cytotoxicity was increased after DOC was encapsulated into polymeric microspheres. In addition, intraperitoneal administration of DOC MS could effectively suppress growth and metastasis of CT26 peritoneal carcinomatosis in vivo, and prolonged the survival of tumor bearing mice. Immunohistochemistry staining of tumor tissues with Ki-67 revealed that DOC MS induced a stronger anti-tumor effect by increasing apoptosis of tumor cells in contrast to other groups (P < 0.05). Thus, our results suggested that DOC MS may have great potential applications in clinic. © 2014 Elsevier B.V. All rights reserved.
1. Introduction In recent years, carrier technology offers an intelligent approach for drug delivery by entrapping the drug into a carrier such as microspheres [1], nanoparticles [2], liposomes [3], and polymer micelles [4] which can improve the efficiency in vivo and reduce toxic side effects of the drug. The microparticulate delivery systems are considered as a potential means to deliver the drug to the target site with specificity, if modified, and to maintain the desired concentration at the site of interest without untoward effects. The use of microsphere-based therapy allows drug release to be carefully tailored to the specific treatment site through the choice and formulation of various drug–polymer combinations. To date, an increasing number of studies have been focused on microspheres loading anticancer drugs [5–8].
∗ Corresponding authors. Tel.: +86 28 85164063; fax: +86 28 85164060. E-mail addresses: luo [email protected] (Y. Luo), [email protected] (G. Guo). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.ijbiomac.2014.05.026 0141-8130/© 2014 Elsevier B.V. All rights reserved.
Anticancer drug docetaxel, a member of the second generation of the taxane family, is obtained by semisynthesis from 10-deacetyl-baccatin III and extracted from the needles of the European Yew Tree Taxus baccata [9,10]. Like paclitaxel, docetaxel exerts its cytotoxic properties by disrupting the function of microtubules and inhibiting the anti-apoptotic gene Bcl2 and encouraging expression of p27, a cell-cycle inhibitor [11,12]. It is about twice as potent as paclitaxel as an inhibitor of microtubule depolymerisation in vitro [13]. Docetaxel has been shown to be a highly potent anticancer agent against various types of cancer, including breast cancer, non-small cell lung cancer, gastric cancer, ovarian cancer, leukemia cancer, prostate cancer, etc. [14,15]. However, from early clinical development of docetaxel, it became clear that docetaxel administration was associated with the occurrence of unpredictable acute hypersensitivity reactions and cumulative fluid retention [16]. Substantial effort has been focused on developing alternative, less toxic and more efficacious formulations for docetaxel [17,18]. Among these, polymeric microspheres are widely studied as controlled drug delivery systems (CDDS) because of their large surface to volume ratios and efficient carrier characteristics [8,19].
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The amphiphilic block copolymer, Pluronic® , consisting of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO) blocks with a PEO–PPO–PEO arrangement (often as EOx–POy–EOx) have been studied for pharmaceutical applications such as solubilization excipients, drug carriers, immunoadjuvants, cryopreservation agents, and gene carriers. Poly(lactic acid)-b-Pluronic-F127 copolymers (PLA-b-F127) [20], Pluronic-F68-b-PLA (F68-PLA) [21], Pluronic P123-b-polyethyleneimine (P123-b-PEI) [22], have been synthesized and frequently applied in CDDS due to their great biocompatibility. And it was reported that Pluronic block copolymer exhibited strong affinity toward small intestines due to the PEO blocks and high permeation characteristic to the cell membrane due to their amphiphilic property [20]. Also, poly(L-lactic acid) (PLA) is a polymer approved by the U.S. Food and Drug Administration for certain clinical applications. At present, PLA is the most popular and widely used synthetic polymeric material, and it has been extensively studied in many fields [23,24]. The block copolymer PLLA–L121–PLLA comprising of commercial Pluronic® L121 (PEO–PPO–PEO) and biodegradable poly(L-lactic acid) (PLLA) was promising for CDDS. Colorectal cancer is one of the leading cancer types and accounts for more than 10% of new cancer cases [25]. Chemotherapy was widely used in the treatment of colorectal cancer and proved to be effective. However, conventional intravenous chemotherapy showed severe systemic toxicity including immune suppression, neurotoxicity, and myelosuppression [26]. This remarkably limited the intensity of chemotherapy and declined the life quality of patients. Intraperitoneal postoperative chemotherapy, which is considered as an adjuvant treatment, was explored to treat the residual tumors remaining after surgery to improve the therapeutic effect [27,28]. To improve the intraperitoneal chemotherapeutic effect, in this study, the block copolymer PLLA–L121–PLLA was synthesized. And then docetaxel-loaded PLLA–L121–PLLA microspheres (DOC MS) for intraperitoneal treatment were prepared by emulsion solvent evaporation method. The particle size, morphological characteristics, encapsulation efficiency, in vitro drug release studies, in vitro cytotoxicity assay and in vivo antitumor activity of DOC MS were examined. Our results suggested that DOC MS may be an efficient and promising protocol for treatment of colorectal peritoneal carcinomatosis. 2. Experimental 2.1. Materials L-lactide was obtained from Guangshui National Chemical Co. PEO–PPO–PEO triblock copolymer (Pluronic® L121) was supplied by BASF (Germany). Polyvinyl alcohol (PVA, average MW 30,000–70,000), docetaxel, stannous octanoate (Sn(Oct)2 ) and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma–Aldrich (USA). Rhodamine B was purchased from Chengdu Ke Long chemicals (China). All other agents like methylbenzene, petroleum ether, dichloromethane, were of analytical reagent (AR) grade and used as received without further purification. 2.2. Synthesis of PLLA–L121–PLLA block copolymer PLLA–Pluronic–PLLA biodegradable block copolymer was synthesized by ring-opening polymerization. Briefly, a known amount of L-lactide and L121 (weight ratio = 10:1) were introduced into a dry glass ampoule under nitrogen atmosphere, and Sn(Oct)2 (0.3 wt.% of L-lactide and L121) was added. The ampoule was kept at 130 ◦ C. During polymerization, the system was stirred
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slowly, and the viscosity increased with time. 10 h later, the system was rapidly heated to 150 ◦ C under vacuum for another 1 h. After cooled to room temperature under nitrogen atmosphere, the PLLA–L121–PLLA block copolymer was first dissolved in methylene chloride and reprecipitated from the filtrate using excess cold petroleum ether. The mixture was filtered and dried to constant weight at 40 ◦ C under vacuum. The obtained purified PLLA–L121–PLLA block copolymer was kept in air-tight bags in desiccators before use. 1 H NMR spectroscopy were recorded on a Varian 400 instrument (Varian, USA) at 400 MHz using deuterated chloroform (CDCl3 ) as the solvent, and tetramethylsilane (TMS) was used as an internal reference standard. 2.2.1. Preparation and characterization of blank PLLA–L121–PLLA, DOC loaded or rhodamine B-loaded microspheres Microspheres containing DOC were formulated by using oil-in-water emulsion solvent evaporation method. Briefly, PLLA–L121–PLLA and DOC were dissolved in 10 ml dichloromethane and added dropwisely to 200 ml of aqueous solution containing PVA (2%, w/v) under stirring with a rotor device (T25, IKA, Germany) at 400 rpm at room temperature. The O/W emulsion formed and was stirred at 200 rpm at room temperature for another 5 h. After the dichloromethane was completely removed, the PLLA–L121–PLLA microspheres formed. Then, the microspheres were washed three times with distilled water. After dispersing in a minimum of water, the microspheres were frozen and lyophilized. The final product was stored until use. Rhodamine B-loaded microspheres were prepared according to the same method described above, and drug-free microspheres were produced in a similar manner without adding the drug. 2.2.2. Characterization of physicochemical property The crystalline states of samples were analyzed on an X’Pert Pro MPD DY1291 (PHILIPS, Netherlands) diffractometer using graphite monochromatized Cu K radiation ( = 0.1542 nm; 40 kV; 40 mA) at a scanning rate of 4◦ /min. The Thermogravimetric analysis (TGA) was taken with a heating rate of 10 ◦ C/min from room-temperature to 600 ◦ C on a TGA Q 500 series Thermogravimetric Analyzer (TA Instrument, USA). 2.2.3. The morphology of microspheres and particle size determination In this paper, Rhodamine B was used as a model fluorescent molecule, which can be encapsulated in the PLLA–L121–PLLA microspheres for qualitative investigation on dispersion of drug in the PLLA–L121–PLLA microspheres. The microspheres were observed by optical microscope. Normal light and polarized light images were acquired at 40× magnification. Scanning electron microscopy (SEM) (JSM-7500F, JEOL, and Japan) was employed to investigate surface morphology of PLLA–L121–PLLA microspheres. The samples were coated with gold before observation. 2.2.4. Drug loading and encapsulation efficiency Reverse-phase high performance liquid chromatography (RPHPLC) was used for determination of DOC. The analysis was carried out at room temperature with an Apollo C18 column (150 mm × 4.6 mm, 5 l; Grace) at 230 nm for DOC. The mixture of acetonitrile and ultrapure water (48:52, v/v) were used as mobile phase and the flow-rate was 1.0 ml/min, respectively. Briefly, exactly weight microspheres were dissolved in dimethyl sulfoxide and the resulting solutions were properly diluted prior to HPLC analysis. The amount of DOC in microspheres was determined from the peak area correlated with the standard curve. All analysis
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was performed in triplicate. Drug loading (DL) and encapsulation efficiency (EE) were calculated according to Eqs. (1) and (2): DL =
drug × 100% polymer + drug
(1)
EE =
experimental drug loading × 100% theoretical drug loading
(2)
2.2.5. In vitro release study In vitro release of DOC from microspheres was carried out in 1 ml of PBS (0.01 M, pH 7.4) containing 0.05% v/v of Tween 80 (to maintain a sink condition) and was placed in a dialysis tube (molecular mass cut off 8–14 kDa). The dialysis tubes was placed in bottles with 20 ml PBS (pH = 7.4, T = 37 ◦ C) in an incubator at 37 ◦ C and kept in a shaker at 120 rpm. At particular time intervals all medium was withdrawn and replaced by equivalent volume of fresh PBS solution at predetermined time points. The concentration of DOC released from DOC MS was determined by HPLC. Three independent experiments were carried out, and the averaged values were used in following data presentation. 2.2.6. In vitro cytotoxicity of blank PLLA–L121–PLLA, free DOC and DOC MS against CT26 colon cancer cells The cytotoxicity of blank MS, DOC MS and free DOC on CT26 cells was evaluation by MTT colorimetric procedure. CT26 cells were plated at a density of 5 × 103 cells per well in 100 l of RPMI 1640 medium in 96-well plates and grown for 24 h. Free DOC was dissolved in DMSO and DOC MS was dispersed in PBS at the concentration of DOC 1 mg/ml and diluted with RPMI 1640 medium to obtain various concentrations of DOC solution. Each cell was washed twice with PBS and incubated with various concentrations of DOC for 1 day. Then the cells were washed with PBS to eliminate the remaining drugs and fresh culture medium was added. MTT solutions were added to per cell and cells were incubated for 4 h at 37 ◦ C. The supernatant was fully removed and 150 l DMSO was added to per cell, oscillating it for 30 min. Then ultraviolet absorbance was measured at 570 nm. The data are expressed as the percentages of viable cells compared to the survival of a control group. 2.3. Animal test In the assays, Balb/c male mice (18–22 g) were used. The animals were maintained at 23 ± 2 ◦ C, food and water were given ad libitum. Four mice groups: Saline group, blank MS group, free DOC group and DOC MS group, each containing eight animals were employed in the treatment effectiveness evaluation of the DOC-
loaded microspheres. The mice were injected with 2 × 105 CT26 cells by intraperitoneal injection. After 7 days, four groups were given different treatments: physiological saline, blank MS solution, free DOC solution and DOC MS solution, respectively. DOC solution was used as a single injection at a dose of 4 mg/kg, and DOC-loaded microspheres solution was used as a single injection at a dose of 8 mg DOC/kg. The free DOC group was treated two times a week and the DOC-loaded microspheres group was given drug once a week. On 14th day of treatment, the mice were sacrificed, and the size and numbers of tumor nodes were measured. 2.4. Immunohistochemical determination of Ki-67 Tumors tissues were harvested, fixed in 4%wt paraformaldehyde (PFA), embedded in paraffin, and sectioned. Ki-67 staining was conducted using the labeled streptavidin–biotin method [29]. The primary antibody and secondary antibody were rat anti-mouse monoclonal antibody Ki-67 (Gene Tech) and biotinylated goat antirat immunoglobulin (BD Biosciences Pharmingen), respectively. To quantify Ki-67 expression, the Ki-67 labeling index (Ki-67 LI) was calculated as number of Ki-67 positive cells/total number of cells counted under 400× magnification in five randomly selected areas in each tumor sample by two independent investigators in a blinded fashion. 2.5. Statistical analysis Quantitative data were expressed as mean ± SD. Statistical comparisons were made by ANOVA analysis and Student’s t-test. P-value < 0.05 was considered statistically significant. 3. Results 3.1. Synthesis and characterization The synthesis scheme of PLLA–L121–PLLA and the schematic illustration of synthesis of PLLA–L121–PLLA microspheres, optical micrographs in normal light or polarized light images were presented in Fig. 1. The optical images polarized light of rhodamine B-loaded microspheres showed highly fluorescent drug was distributed homogenously in the beads. 1 H NMR spectra of PLLA–L121–PLLA was recorded and shown in Fig. 2. The characteristic absorption peaks were also indicated in this figure. The characteristic absorption peaks were also designated. In Fig. 2, peak at 5.20 ppm belonged to the methane proton of PLLA blocks (O–CH(CH3 )–CO–, peaks at ı = 3.35–3.70 belonged to the methyl protons of L121 block (–OCH2 –CH2 – and
Fig. 1. Preparation and fluorescence studies of PLLA–L121–PLLA microspheres. (A) Synthesis scheme of PLLA–L121–PLLA; (B) preparation scheme of DOC MS (a: the mix CH2 Cl2 solution of PLLA–L121–PLLA and DOC; b: dispersed phase in 2% PVA solution); (C) bright field of rhodamine B MS; (D) fuorescence field of rhodamine B MS.
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Table 1 DOC MS prepared in this work. All data were expressed as the mean ± SD (n = 3).
Fig. 2.
1
H NMR spectrums of PLLA–L121–PLLA block copolymer.
Sample
Theoretical drug loading (%)
DL (%)
EL (%)
Size (m)
MS-1 MS-2 MS-3 MS-4 MS-5
0 5 10 15 20
0 3.68 ± 0.29 7.09 ± 0.42 9.92 ± 1.26 10.98 ± 2.27
0 73.58 ± 5.57 70.68 ± 4.18 66.24 ± 6.29 54.86 ± 11.36
36.34 40.46 45.85 54.85 55.09
± ± ± ± ±
2.56 1.48 1.65 2.76 3.09
could be divided into two stages. In Fig. 4b, it can be seen that the tendency of thermal degradation curves of DOC MS did not show obviously difference except the residue at 600 ◦ C. And the residue at 600 ◦ C increased when the theoretical drug loading of DOC MS increased from 5% to 10%. This may suggested that the DOC was successfully loaded in the microspheres. 3.2. Morphology of DOC-loaded microspheres
Fig. 3. XRD patterns of pure DOC (A), PLLA–L121–PLLA (B), the mixture of DOC and PLLA–L121–PLLA (C) and DOC MS (D).
DOC MS was prepared by an O/W emulsion solvent evaporation method, drug loading efficiency, size of the microspheres samples were listed along with the drug loading in Table 1. As shown in Table 1, when theoretical drug loading increased from 0 to 30, particle size and drug loading both increased accordingly, whereas encapsulation efficiency decreased slightly. For the consideration of particle size and drug loading, MS-3 was used in the following in vitro and in vivo studies. Fig. 5 showed SEM photographs of blank MS and DOC MS, the surface of the microspheres was porous. Both optical and SEM micrographs showed that drug-loaded microspheres were spherical. The optical images polarized light of drug-loaded microspheres showed highly fluorescent drug was distributed homogenously in the beads. 3.3. In vitro drug release studies
–OCH2 –CH(CH3 )–. The small peak at ı = 4.3–4.4 ppm belonged to the methylene protons of PLLA–CO–OCH2 –CH2 –O–L121– segment. Fig. 3 presented the X-ray diffraction patterns of pure DOC (A), PLLA–L121–PLLA (B), the mixture of DOC and PLLA–L121–PLLA (C) and DOC MS (D). Pure DOC is crystalline, with characteristic peaks at 2 = 8.0◦ , 9.2◦ , 11.3◦ , 12.5◦ , 13.8◦ , and 16.9◦ respectively. In comparison with XRD diagram of DOC, blank MS and physical mixtures of DOC and PLLA–L121–PLLA (10%) and DOC MS (DL = 8%) freezedried powder, the absence of specific diffraction peaks in diagram of DOC MS indicated that DOC was encapsulated amorphously. Thermogravimetric curves of blank MS, DOC MS 5%, DOC MS 10% and pure DOC were showed in Fig. 4. The results revealed that the thermal degradation of the PLLA–L121–PLLA block copolymer
Fig. 6 showed the release profiles of free DOC and DOC MS microspheres in PBS (pH 7.4, 37 ◦ C). In comparison to free DOC, a typical two-phase-release profile of DOC MS was observed. That is, a relatively rapid release in the first stage followed by a sustained and slow release over a prolonged time up to several weeks. It was found that only 17% DOC released from DOC MS within 24 h, while free DOC released about 80% into the outside media. 3.4. The cytotoxicity of DOC-loaded microspheres The MTT assay was performed to evaluate the toxicity of drug-loaded polymeric microspheres and free DOC to investigate whether microspheres influenced the cytotoxity of honokiol. Both free DOC and DOC-loaded microspheres at various concentrations significantly decreased the viability of CT26 cells in a dose-dependent manner. Fig. 7 showed the influence of drug loading on cell viability. The result indicated that the cytotoxicity of DOC-loaded microspheres is comparable to that of free DOC. 3.5. Animal test
Fig. 4. Thermogravimetric curves of blank MS, DOC MS 5%, DOC MS 10% and pure DOC.
Abdominal metastases of CT26 colon carcinom a model was established and used to evaluate the therapeutic effectiveness. Thirty-two mice were divided four groups treated with physiological saline, blank MS, free DOC and DOC MS (DOC content was 6.3%). The anticancer activity of the intraperitoneal injection of DOC-loaded microspheres in Balb/c mice bearing peritoneal carcinomatosis of CT26 colon carcinoma was illustrated in Fig. 8. The images of abdominal cavity showed that the tumor node numbers from DOC-loaded microspheres treated group were significantly
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Fig. 5. Scanning electron micrographs of microspheres: blank MS (A 400×, B 800×); DOC MS (C 400×, D 800×).
less than other groups. Furthermore, the size of tumor nodes was significantly smaller in comparison to other groups. The in vivo survival rates of free DOC and DOC MS were also examined (Fig. 8D). After administration of saline and blank microspheres, all rats died
within 25 days with a median survival of 23 and 25 days respectively, due to the rapid growth of tumors. The median survival in DOC MS group (33 days) is significantly longer compared with free DOC (29 days, P < 0.05), blank MS (25 days, P < 0.05), and saline (23 days, P < 0.05) group. 3.6. Determination of tumor cell proliferation To evaluate the effect of DOC-loaded microspheres on proliferation of tumor, immunohistochemical staining murine Ki-67 was carried out. According to Fig. 9A–D, within a similar high-power field, weak Ki-67 immunoreactivity in tumor tissues were observed in DOC MS treated mice compared with those in free DOC, blank MS, or saline group. The Ki-67 LI of tumor tissues was significantly lower in DOC MS group (25.46 ± 4.65%) than in free DOC (43.12 ± 3.54%, P < 0.001), blank MS (73.16 ± 5.34%, P < 0.001), or saline group (78.98 ± 4.28%, P < 0.001), respectively (Fig. 9E). These indicated that DOC MS could induce tumor cell apoptosis, inhibit tumor angiogenesis, and suppressed tumor cell proliferation.
Fig. 6. In vitro release behavior of DOC from free DOC and DOC MS. Error bars represent the SD (n = 3).
4. Discussion
Fig. 7. In vitro cytotoxicity of blank MS, free DOC and DOC MS against CT26 cells confirmed by MTT assay according to the DOC concentration.
With the advent of biocompatible and biodegradable polymers, much research has focused on the development of suitable polymeric drug delivery systems and their design for sustained drug release applications [30–32]. Among this, biodegradable polymer microspheres have been widely used because of their sustained drug action on the lesion, high capability to cross various physiological barriers, controlled and targeted delivery of the drugs. Moreover, they offer facile administration via routes including oral, pulmonary and parenteral injection, and they do not need surgical removal upon complete drug release [33,34]. In particular, encapsulation of docetaxel in microspheres has been investigated over the past decades because docetaxel has poor water solubility, high toxicity and low bioavailability [35,36]. In its clinical use, tween 80 and ethanol are often added as solubiliser, which lead to some unpredictable toxicity such as hypersensitivity, cumulativefluid retention, neurotoxicity, diarrhea and the most commonly
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Fig. 8. Inraperitoneal administration of DOC MS inhibited the growth of abdominal metastases of CT26 colon carcinoma. (A) Representative photographs of abdominal metastatic nodes in each treatment group; (B) mean number of tumor nodes in each group; (C) the weight of mice in each group; (D) survival curve of mice in each group.
neutropenia [37]. It became more and more important to change formulation of docetaxel in order to achieve better therapeutic effects and reduce the side-effect of docetaxel for its tumor therapy application. In previous studies, Pluronic/poly(lactic acid) block copolymers with different structural compositions and properties have been synthesized for several applications, including drug delivery systems. Xiong et al. [38] successfully grafted poly(lactic acid) to both ends of Pluronic F127 block copolymers to obtain amphiphilic PLA–F127–PLA block copolymers. The results suggested that the permeability and chain mobility of PLA blocks of PLA–F127–PLA block polymers in water were increased and the release of drugs from PLA–F127–PLA nanoparticles is mainly controlled by concentration gradient due to the slow hydrolytic degradation of PLA segments. Mu et al. [39] developed a mixed polymeric micellar formulation comprised of methoxy poly(ethylene glycol)–poly (lactide) polymer and Pluronic® triblock polymers for enhanced bioavailability and to overcome MDR of docetaxel. However, there is no literature about Pluronic L121 used in microspheres for drug delivery according to our knowledge. In this work, we developed a system of PLLA–L121–PLLA microspheres for sustained and controlled delivery of DOC, and then several observations were made concerning the preparation and therapeutic effect of DOC MS on CT26 colon carcinoma in vitro and in vivo. 1 H NMR showed that PLLA–L121–PLLA block copolymer was successfully synthesized. The X-ray diffraction analysis revealed the micro-domain structure of docetaxel in the polymeric microspheres. The spectrum showed a transformation of docetaxel from crystalline structure to amorphous microspheres. The average diameter of DOC-loaded microspheres was about 45 m. This, in combination with the surface morphology of microspheres suggested that the prepared docetaxel porous microspheres had uniform particle size, stable drug loading and sustained drug release behavior by the emulsification and solvent evaporation technique.
Compared with the rapid clearance of nanoparticles or micelles from peritoneal cavity, retention time of microspheres is much longer and peritoneal concentration is higher [40]. The much slower release of docetaxel from PLLA–L121–PLLA microspheres in comparison with free docetaxel can be attributed to the molecular structural characteristics of polymeric microspheres. This delay of drug release indicates their potential applicability in drug carrier to minimize the exposure of healthy tissues while increasing the accumulation of therapeutic drug in the tumor site. Moreover, cytotoxicity results revealed that the DOC-loaded microspheres remained the comparable anticancer effect even though docetaxel was released in an extended behavior. Pluronic® block copolymers were used in the study for its commercial availability, biocompatibility and safety [41]. Moreover, studies have reported that Pluronic block copolymers sensitized the resistant cancer cell lines, so that the cytotoxic activity of the drug could increase by 2–3 orders of magnitude [42]. The interstitial chemotherapy by local injection of sustainedrelease medication has many advantages, including minimal invasion, high drug concentration in the injection local, and lowered systemic toxicity, etc. The results of DOC-loaded microspheres chemotherapy showed significant anticancer effect on the mice bearing colorectal peritoneal carcinomatosis. It is considered that the increased survival rate of mice treated with DOC-loaded microspheres is probably due to the better antitumor efficacies of these particles. PLLA–L121–PLLA microspheres supplied active DOC molecules for extended times through a sustained release pattern, thereby exerting an antitumor effect in the CT26 colon carcinoma. Immunohistochemical staining with Ki-67 monoclonal antibody is a frequently used method for evaluation of cell proliferation. Sections of tumors from mice in each group were stained for Ki67 to determine the proliferation of tumor cells. According to Fig. 9A–D, weak Ki-67 immunoreactivity observed in tumor tissue from mice treated with DOC MS in comparison with free DOC, blank MS or NS. In Fig. 9E, Ki-67 LI of tumor tissues from DOC MS treated mice was significant lower compared with free DOC, blank MS, or
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Fig. 9. Ki-67 immunohistochemical staining of tumors. Representative Ki-67 immunohistochemical images of saline (A), blank MS (B), free DOC (C), and DOC MS (D) group, and mean Ki-67 LI in each group (E).
NS group. Results of Ki-67 staining of tumor tissues suggested that DOC MS could suppress tumor cell proliferation. The local injection of DOC-loaded microspheres demonstrated a significant antiangiogenic effect in our study. 5. Conclusion Biodegradable DOC MS were prepared and assigned for CRPC therapy. In vitro and in vivo toxicity evaluation indicated that after DOC was encapsulated into polymeric microspheres, in vitro cytotoxicity was increased compared with free DOC. Besides, a sustained in vitro release behavior was also observed in DOC MS group. Furthermore, compared with free DOC, intraperitoneal administration of DOC MS was more effective in suppressing tumor growth and metastasis and prolonged survival in vivo. Encapsulation of DOC in polymeric microspheres increased concentration and retention time in plasma and tumors. The PLLA–L121–PLLA block copolymers possess excellent biocompatibility because of the Pluronic and PLLA segments. In addition, the biodegradability of PLLA blocks makes it attractive for use in controlled drug release systems because the release rates can be controlled by varying the length and degradation rate of PLLA blocks. Due to its effectiveness and safety, the
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Docetaxel load biodegradable porous microspheres for the treatment of colorectal peritoneal carcinomatosis RangRang Fan a,1 , YueLong Wang a,1 , Bo Han b , YouFu Luo a,∗ , LiangXue Zhou a , XiRui Peng a , Min Wu a , Yu Zheng a , Gang Guo a,∗ a State Key Laboratory of Biotherapy and Cancer Center, and Department of Neurosurgery, West China Hospital, West China Medical School, Sichuan University, Chengdu 610041, PR China b Key Laboratory of Xinjiang Phytomedicine Resources, Shihezi 832002, PR China
a r t i c l e
i n f o
Article history: Received 16 March 2014 Received in revised form 20 April 2014 Accepted 7 May 2014 Available online 20 May 2014 Keywords: PLLA–L121–PLLA Porous microspheres Colorectal peritoneal carcinomatosis
a b s t r a c t Micro- and nanoparticle formulations are widely used to improve the bioavailability of low solubility drugs. In this study, biodegradable poly(L-lactide acid)–Pluronic L121–poly(L-lactide acid) (PLLA–L121–PLLA) was developed. And then a controlled drug delivery system (CDDS), docetaxel (DOC) loaded PLLA–L121–PLLA porous microsphere (DOC MS) was prepared for colorectal peritoneal carcinomatosis (CRPC) therapy. DOC MS was prepared by DOC and PLLA–L121–PLLA using an oil-in-water emulsion solvent evaporation method. The particle size, morphological characteristics, encapsulation efficiency, in vitro drug release studies and in vitro cytotoxicity of DOC MS have been investigated. In vitro release profile demonstrated a significant difference between rapid release of free DOC and much slower and sustained release of DOC MS. Furthermore, cytotoxicity assay indicated cytotoxicity was increased after DOC was encapsulated into polymeric microspheres. In addition, intraperitoneal administration of DOC MS could effectively suppress growth and metastasis of CT26 peritoneal carcinomatosis in vivo, and prolonged the survival of tumor bearing mice. Immunohistochemistry staining of tumor tissues with Ki-67 revealed that DOC MS induced a stronger anti-tumor effect by increasing apoptosis of tumor cells in contrast to other groups (P < 0.05). Thus, our results suggested that DOC MS may have great potential applications in clinic. © 2014 Elsevier B.V. All rights reserved.
1. Introduction In recent years, carrier technology offers an intelligent approach for drug delivery by entrapping the drug into a carrier such as microspheres [1], nanoparticles [2], liposomes [3], and polymer micelles [4] which can improve the efficiency in vivo and reduce toxic side effects of the drug. The microparticulate delivery systems are considered as a potential means to deliver the drug to the target site with specificity, if modified, and to maintain the desired concentration at the site of interest without untoward effects. The use of microsphere-based therapy allows drug release to be carefully tailored to the specific treatment site through the choice and formulation of various drug–polymer combinations. To date, an increasing number of studies have been focused on microspheres loading anticancer drugs [5–8].
∗ Corresponding authors. Tel.: +86 28 85164063; fax: +86 28 85164060. E-mail addresses: luo [email protected] (Y. Luo), [email protected] (G. Guo). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.ijbiomac.2014.05.026 0141-8130/© 2014 Elsevier B.V. All rights reserved.
Anticancer drug docetaxel, a member of the second generation of the taxane family, is obtained by semisynthesis from 10-deacetyl-baccatin III and extracted from the needles of the European Yew Tree Taxus baccata [9,10]. Like paclitaxel, docetaxel exerts its cytotoxic properties by disrupting the function of microtubules and inhibiting the anti-apoptotic gene Bcl2 and encouraging expression of p27, a cell-cycle inhibitor [11,12]. It is about twice as potent as paclitaxel as an inhibitor of microtubule depolymerisation in vitro [13]. Docetaxel has been shown to be a highly potent anticancer agent against various types of cancer, including breast cancer, non-small cell lung cancer, gastric cancer, ovarian cancer, leukemia cancer, prostate cancer, etc. [14,15]. However, from early clinical development of docetaxel, it became clear that docetaxel administration was associated with the occurrence of unpredictable acute hypersensitivity reactions and cumulative fluid retention [16]. Substantial effort has been focused on developing alternative, less toxic and more efficacious formulations for docetaxel [17,18]. Among these, polymeric microspheres are widely studied as controlled drug delivery systems (CDDS) because of their large surface to volume ratios and efficient carrier characteristics [8,19].
R. Fan et al. / International Journal of Biological Macromolecules 69 (2014) 100–107
The amphiphilic block copolymer, Pluronic® , consisting of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO) blocks with a PEO–PPO–PEO arrangement (often as EOx–POy–EOx) have been studied for pharmaceutical applications such as solubilization excipients, drug carriers, immunoadjuvants, cryopreservation agents, and gene carriers. Poly(lactic acid)-b-Pluronic-F127 copolymers (PLA-b-F127) [20], Pluronic-F68-b-PLA (F68-PLA) [21], Pluronic P123-b-polyethyleneimine (P123-b-PEI) [22], have been synthesized and frequently applied in CDDS due to their great biocompatibility. And it was reported that Pluronic block copolymer exhibited strong affinity toward small intestines due to the PEO blocks and high permeation characteristic to the cell membrane due to their amphiphilic property [20]. Also, poly(L-lactic acid) (PLA) is a polymer approved by the U.S. Food and Drug Administration for certain clinical applications. At present, PLA is the most popular and widely used synthetic polymeric material, and it has been extensively studied in many fields [23,24]. The block copolymer PLLA–L121–PLLA comprising of commercial Pluronic® L121 (PEO–PPO–PEO) and biodegradable poly(L-lactic acid) (PLLA) was promising for CDDS. Colorectal cancer is one of the leading cancer types and accounts for more than 10% of new cancer cases [25]. Chemotherapy was widely used in the treatment of colorectal cancer and proved to be effective. However, conventional intravenous chemotherapy showed severe systemic toxicity including immune suppression, neurotoxicity, and myelosuppression [26]. This remarkably limited the intensity of chemotherapy and declined the life quality of patients. Intraperitoneal postoperative chemotherapy, which is considered as an adjuvant treatment, was explored to treat the residual tumors remaining after surgery to improve the therapeutic effect [27,28]. To improve the intraperitoneal chemotherapeutic effect, in this study, the block copolymer PLLA–L121–PLLA was synthesized. And then docetaxel-loaded PLLA–L121–PLLA microspheres (DOC MS) for intraperitoneal treatment were prepared by emulsion solvent evaporation method. The particle size, morphological characteristics, encapsulation efficiency, in vitro drug release studies, in vitro cytotoxicity assay and in vivo antitumor activity of DOC MS were examined. Our results suggested that DOC MS may be an efficient and promising protocol for treatment of colorectal peritoneal carcinomatosis. 2. Experimental 2.1. Materials L-lactide was obtained from Guangshui National Chemical Co. PEO–PPO–PEO triblock copolymer (Pluronic® L121) was supplied by BASF (Germany). Polyvinyl alcohol (PVA, average MW 30,000–70,000), docetaxel, stannous octanoate (Sn(Oct)2 ) and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma–Aldrich (USA). Rhodamine B was purchased from Chengdu Ke Long chemicals (China). All other agents like methylbenzene, petroleum ether, dichloromethane, were of analytical reagent (AR) grade and used as received without further purification. 2.2. Synthesis of PLLA–L121–PLLA block copolymer PLLA–Pluronic–PLLA biodegradable block copolymer was synthesized by ring-opening polymerization. Briefly, a known amount of L-lactide and L121 (weight ratio = 10:1) were introduced into a dry glass ampoule under nitrogen atmosphere, and Sn(Oct)2 (0.3 wt.% of L-lactide and L121) was added. The ampoule was kept at 130 ◦ C. During polymerization, the system was stirred
101
slowly, and the viscosity increased with time. 10 h later, the system was rapidly heated to 150 ◦ C under vacuum for another 1 h. After cooled to room temperature under nitrogen atmosphere, the PLLA–L121–PLLA block copolymer was first dissolved in methylene chloride and reprecipitated from the filtrate using excess cold petroleum ether. The mixture was filtered and dried to constant weight at 40 ◦ C under vacuum. The obtained purified PLLA–L121–PLLA block copolymer was kept in air-tight bags in desiccators before use. 1 H NMR spectroscopy were recorded on a Varian 400 instrument (Varian, USA) at 400 MHz using deuterated chloroform (CDCl3 ) as the solvent, and tetramethylsilane (TMS) was used as an internal reference standard. 2.2.1. Preparation and characterization of blank PLLA–L121–PLLA, DOC loaded or rhodamine B-loaded microspheres Microspheres containing DOC were formulated by using oil-in-water emulsion solvent evaporation method. Briefly, PLLA–L121–PLLA and DOC were dissolved in 10 ml dichloromethane and added dropwisely to 200 ml of aqueous solution containing PVA (2%, w/v) under stirring with a rotor device (T25, IKA, Germany) at 400 rpm at room temperature. The O/W emulsion formed and was stirred at 200 rpm at room temperature for another 5 h. After the dichloromethane was completely removed, the PLLA–L121–PLLA microspheres formed. Then, the microspheres were washed three times with distilled water. After dispersing in a minimum of water, the microspheres were frozen and lyophilized. The final product was stored until use. Rhodamine B-loaded microspheres were prepared according to the same method described above, and drug-free microspheres were produced in a similar manner without adding the drug. 2.2.2. Characterization of physicochemical property The crystalline states of samples were analyzed on an X’Pert Pro MPD DY1291 (PHILIPS, Netherlands) diffractometer using graphite monochromatized Cu K radiation ( = 0.1542 nm; 40 kV; 40 mA) at a scanning rate of 4◦ /min. The Thermogravimetric analysis (TGA) was taken with a heating rate of 10 ◦ C/min from room-temperature to 600 ◦ C on a TGA Q 500 series Thermogravimetric Analyzer (TA Instrument, USA). 2.2.3. The morphology of microspheres and particle size determination In this paper, Rhodamine B was used as a model fluorescent molecule, which can be encapsulated in the PLLA–L121–PLLA microspheres for qualitative investigation on dispersion of drug in the PLLA–L121–PLLA microspheres. The microspheres were observed by optical microscope. Normal light and polarized light images were acquired at 40× magnification. Scanning electron microscopy (SEM) (JSM-7500F, JEOL, and Japan) was employed to investigate surface morphology of PLLA–L121–PLLA microspheres. The samples were coated with gold before observation. 2.2.4. Drug loading and encapsulation efficiency Reverse-phase high performance liquid chromatography (RPHPLC) was used for determination of DOC. The analysis was carried out at room temperature with an Apollo C18 column (150 mm × 4.6 mm, 5 l; Grace) at 230 nm for DOC. The mixture of acetonitrile and ultrapure water (48:52, v/v) were used as mobile phase and the flow-rate was 1.0 ml/min, respectively. Briefly, exactly weight microspheres were dissolved in dimethyl sulfoxide and the resulting solutions were properly diluted prior to HPLC analysis. The amount of DOC in microspheres was determined from the peak area correlated with the standard curve. All analysis
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was performed in triplicate. Drug loading (DL) and encapsulation efficiency (EE) were calculated according to Eqs. (1) and (2): DL =
drug × 100% polymer + drug
(1)
EE =
experimental drug loading × 100% theoretical drug loading
(2)
2.2.5. In vitro release study In vitro release of DOC from microspheres was carried out in 1 ml of PBS (0.01 M, pH 7.4) containing 0.05% v/v of Tween 80 (to maintain a sink condition) and was placed in a dialysis tube (molecular mass cut off 8–14 kDa). The dialysis tubes was placed in bottles with 20 ml PBS (pH = 7.4, T = 37 ◦ C) in an incubator at 37 ◦ C and kept in a shaker at 120 rpm. At particular time intervals all medium was withdrawn and replaced by equivalent volume of fresh PBS solution at predetermined time points. The concentration of DOC released from DOC MS was determined by HPLC. Three independent experiments were carried out, and the averaged values were used in following data presentation. 2.2.6. In vitro cytotoxicity of blank PLLA–L121–PLLA, free DOC and DOC MS against CT26 colon cancer cells The cytotoxicity of blank MS, DOC MS and free DOC on CT26 cells was evaluation by MTT colorimetric procedure. CT26 cells were plated at a density of 5 × 103 cells per well in 100 l of RPMI 1640 medium in 96-well plates and grown for 24 h. Free DOC was dissolved in DMSO and DOC MS was dispersed in PBS at the concentration of DOC 1 mg/ml and diluted with RPMI 1640 medium to obtain various concentrations of DOC solution. Each cell was washed twice with PBS and incubated with various concentrations of DOC for 1 day. Then the cells were washed with PBS to eliminate the remaining drugs and fresh culture medium was added. MTT solutions were added to per cell and cells were incubated for 4 h at 37 ◦ C. The supernatant was fully removed and 150 l DMSO was added to per cell, oscillating it for 30 min. Then ultraviolet absorbance was measured at 570 nm. The data are expressed as the percentages of viable cells compared to the survival of a control group. 2.3. Animal test In the assays, Balb/c male mice (18–22 g) were used. The animals were maintained at 23 ± 2 ◦ C, food and water were given ad libitum. Four mice groups: Saline group, blank MS group, free DOC group and DOC MS group, each containing eight animals were employed in the treatment effectiveness evaluation of the DOC-
loaded microspheres. The mice were injected with 2 × 105 CT26 cells by intraperitoneal injection. After 7 days, four groups were given different treatments: physiological saline, blank MS solution, free DOC solution and DOC MS solution, respectively. DOC solution was used as a single injection at a dose of 4 mg/kg, and DOC-loaded microspheres solution was used as a single injection at a dose of 8 mg DOC/kg. The free DOC group was treated two times a week and the DOC-loaded microspheres group was given drug once a week. On 14th day of treatment, the mice were sacrificed, and the size and numbers of tumor nodes were measured. 2.4. Immunohistochemical determination of Ki-67 Tumors tissues were harvested, fixed in 4%wt paraformaldehyde (PFA), embedded in paraffin, and sectioned. Ki-67 staining was conducted using the labeled streptavidin–biotin method [29]. The primary antibody and secondary antibody were rat anti-mouse monoclonal antibody Ki-67 (Gene Tech) and biotinylated goat antirat immunoglobulin (BD Biosciences Pharmingen), respectively. To quantify Ki-67 expression, the Ki-67 labeling index (Ki-67 LI) was calculated as number of Ki-67 positive cells/total number of cells counted under 400× magnification in five randomly selected areas in each tumor sample by two independent investigators in a blinded fashion. 2.5. Statistical analysis Quantitative data were expressed as mean ± SD. Statistical comparisons were made by ANOVA analysis and Student’s t-test. P-value < 0.05 was considered statistically significant. 3. Results 3.1. Synthesis and characterization The synthesis scheme of PLLA–L121–PLLA and the schematic illustration of synthesis of PLLA–L121–PLLA microspheres, optical micrographs in normal light or polarized light images were presented in Fig. 1. The optical images polarized light of rhodamine B-loaded microspheres showed highly fluorescent drug was distributed homogenously in the beads. 1 H NMR spectra of PLLA–L121–PLLA was recorded and shown in Fig. 2. The characteristic absorption peaks were also indicated in this figure. The characteristic absorption peaks were also designated. In Fig. 2, peak at 5.20 ppm belonged to the methane proton of PLLA blocks (O–CH(CH3 )–CO–, peaks at ı = 3.35–3.70 belonged to the methyl protons of L121 block (–OCH2 –CH2 – and
Fig. 1. Preparation and fluorescence studies of PLLA–L121–PLLA microspheres. (A) Synthesis scheme of PLLA–L121–PLLA; (B) preparation scheme of DOC MS (a: the mix CH2 Cl2 solution of PLLA–L121–PLLA and DOC; b: dispersed phase in 2% PVA solution); (C) bright field of rhodamine B MS; (D) fuorescence field of rhodamine B MS.
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Table 1 DOC MS prepared in this work. All data were expressed as the mean ± SD (n = 3).
Fig. 2.
1
H NMR spectrums of PLLA–L121–PLLA block copolymer.
Sample
Theoretical drug loading (%)
DL (%)
EL (%)
Size (m)
MS-1 MS-2 MS-3 MS-4 MS-5
0 5 10 15 20
0 3.68 ± 0.29 7.09 ± 0.42 9.92 ± 1.26 10.98 ± 2.27
0 73.58 ± 5.57 70.68 ± 4.18 66.24 ± 6.29 54.86 ± 11.36
36.34 40.46 45.85 54.85 55.09
± ± ± ± ±
2.56 1.48 1.65 2.76 3.09
could be divided into two stages. In Fig. 4b, it can be seen that the tendency of thermal degradation curves of DOC MS did not show obviously difference except the residue at 600 ◦ C. And the residue at 600 ◦ C increased when the theoretical drug loading of DOC MS increased from 5% to 10%. This may suggested that the DOC was successfully loaded in the microspheres. 3.2. Morphology of DOC-loaded microspheres
Fig. 3. XRD patterns of pure DOC (A), PLLA–L121–PLLA (B), the mixture of DOC and PLLA–L121–PLLA (C) and DOC MS (D).
DOC MS was prepared by an O/W emulsion solvent evaporation method, drug loading efficiency, size of the microspheres samples were listed along with the drug loading in Table 1. As shown in Table 1, when theoretical drug loading increased from 0 to 30, particle size and drug loading both increased accordingly, whereas encapsulation efficiency decreased slightly. For the consideration of particle size and drug loading, MS-3 was used in the following in vitro and in vivo studies. Fig. 5 showed SEM photographs of blank MS and DOC MS, the surface of the microspheres was porous. Both optical and SEM micrographs showed that drug-loaded microspheres were spherical. The optical images polarized light of drug-loaded microspheres showed highly fluorescent drug was distributed homogenously in the beads. 3.3. In vitro drug release studies
–OCH2 –CH(CH3 )–. The small peak at ı = 4.3–4.4 ppm belonged to the methylene protons of PLLA–CO–OCH2 –CH2 –O–L121– segment. Fig. 3 presented the X-ray diffraction patterns of pure DOC (A), PLLA–L121–PLLA (B), the mixture of DOC and PLLA–L121–PLLA (C) and DOC MS (D). Pure DOC is crystalline, with characteristic peaks at 2 = 8.0◦ , 9.2◦ , 11.3◦ , 12.5◦ , 13.8◦ , and 16.9◦ respectively. In comparison with XRD diagram of DOC, blank MS and physical mixtures of DOC and PLLA–L121–PLLA (10%) and DOC MS (DL = 8%) freezedried powder, the absence of specific diffraction peaks in diagram of DOC MS indicated that DOC was encapsulated amorphously. Thermogravimetric curves of blank MS, DOC MS 5%, DOC MS 10% and pure DOC were showed in Fig. 4. The results revealed that the thermal degradation of the PLLA–L121–PLLA block copolymer
Fig. 6 showed the release profiles of free DOC and DOC MS microspheres in PBS (pH 7.4, 37 ◦ C). In comparison to free DOC, a typical two-phase-release profile of DOC MS was observed. That is, a relatively rapid release in the first stage followed by a sustained and slow release over a prolonged time up to several weeks. It was found that only 17% DOC released from DOC MS within 24 h, while free DOC released about 80% into the outside media. 3.4. The cytotoxicity of DOC-loaded microspheres The MTT assay was performed to evaluate the toxicity of drug-loaded polymeric microspheres and free DOC to investigate whether microspheres influenced the cytotoxity of honokiol. Both free DOC and DOC-loaded microspheres at various concentrations significantly decreased the viability of CT26 cells in a dose-dependent manner. Fig. 7 showed the influence of drug loading on cell viability. The result indicated that the cytotoxicity of DOC-loaded microspheres is comparable to that of free DOC. 3.5. Animal test
Fig. 4. Thermogravimetric curves of blank MS, DOC MS 5%, DOC MS 10% and pure DOC.
Abdominal metastases of CT26 colon carcinom a model was established and used to evaluate the therapeutic effectiveness. Thirty-two mice were divided four groups treated with physiological saline, blank MS, free DOC and DOC MS (DOC content was 6.3%). The anticancer activity of the intraperitoneal injection of DOC-loaded microspheres in Balb/c mice bearing peritoneal carcinomatosis of CT26 colon carcinoma was illustrated in Fig. 8. The images of abdominal cavity showed that the tumor node numbers from DOC-loaded microspheres treated group were significantly
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Fig. 5. Scanning electron micrographs of microspheres: blank MS (A 400×, B 800×); DOC MS (C 400×, D 800×).
less than other groups. Furthermore, the size of tumor nodes was significantly smaller in comparison to other groups. The in vivo survival rates of free DOC and DOC MS were also examined (Fig. 8D). After administration of saline and blank microspheres, all rats died
within 25 days with a median survival of 23 and 25 days respectively, due to the rapid growth of tumors. The median survival in DOC MS group (33 days) is significantly longer compared with free DOC (29 days, P < 0.05), blank MS (25 days, P < 0.05), and saline (23 days, P < 0.05) group. 3.6. Determination of tumor cell proliferation To evaluate the effect of DOC-loaded microspheres on proliferation of tumor, immunohistochemical staining murine Ki-67 was carried out. According to Fig. 9A–D, within a similar high-power field, weak Ki-67 immunoreactivity in tumor tissues were observed in DOC MS treated mice compared with those in free DOC, blank MS, or saline group. The Ki-67 LI of tumor tissues was significantly lower in DOC MS group (25.46 ± 4.65%) than in free DOC (43.12 ± 3.54%, P < 0.001), blank MS (73.16 ± 5.34%, P < 0.001), or saline group (78.98 ± 4.28%, P < 0.001), respectively (Fig. 9E). These indicated that DOC MS could induce tumor cell apoptosis, inhibit tumor angiogenesis, and suppressed tumor cell proliferation.
Fig. 6. In vitro release behavior of DOC from free DOC and DOC MS. Error bars represent the SD (n = 3).
4. Discussion
Fig. 7. In vitro cytotoxicity of blank MS, free DOC and DOC MS against CT26 cells confirmed by MTT assay according to the DOC concentration.
With the advent of biocompatible and biodegradable polymers, much research has focused on the development of suitable polymeric drug delivery systems and their design for sustained drug release applications [30–32]. Among this, biodegradable polymer microspheres have been widely used because of their sustained drug action on the lesion, high capability to cross various physiological barriers, controlled and targeted delivery of the drugs. Moreover, they offer facile administration via routes including oral, pulmonary and parenteral injection, and they do not need surgical removal upon complete drug release [33,34]. In particular, encapsulation of docetaxel in microspheres has been investigated over the past decades because docetaxel has poor water solubility, high toxicity and low bioavailability [35,36]. In its clinical use, tween 80 and ethanol are often added as solubiliser, which lead to some unpredictable toxicity such as hypersensitivity, cumulativefluid retention, neurotoxicity, diarrhea and the most commonly
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Fig. 8. Inraperitoneal administration of DOC MS inhibited the growth of abdominal metastases of CT26 colon carcinoma. (A) Representative photographs of abdominal metastatic nodes in each treatment group; (B) mean number of tumor nodes in each group; (C) the weight of mice in each group; (D) survival curve of mice in each group.
neutropenia [37]. It became more and more important to change formulation of docetaxel in order to achieve better therapeutic effects and reduce the side-effect of docetaxel for its tumor therapy application. In previous studies, Pluronic/poly(lactic acid) block copolymers with different structural compositions and properties have been synthesized for several applications, including drug delivery systems. Xiong et al. [38] successfully grafted poly(lactic acid) to both ends of Pluronic F127 block copolymers to obtain amphiphilic PLA–F127–PLA block copolymers. The results suggested that the permeability and chain mobility of PLA blocks of PLA–F127–PLA block polymers in water were increased and the release of drugs from PLA–F127–PLA nanoparticles is mainly controlled by concentration gradient due to the slow hydrolytic degradation of PLA segments. Mu et al. [39] developed a mixed polymeric micellar formulation comprised of methoxy poly(ethylene glycol)–poly (lactide) polymer and Pluronic® triblock polymers for enhanced bioavailability and to overcome MDR of docetaxel. However, there is no literature about Pluronic L121 used in microspheres for drug delivery according to our knowledge. In this work, we developed a system of PLLA–L121–PLLA microspheres for sustained and controlled delivery of DOC, and then several observations were made concerning the preparation and therapeutic effect of DOC MS on CT26 colon carcinoma in vitro and in vivo. 1 H NMR showed that PLLA–L121–PLLA block copolymer was successfully synthesized. The X-ray diffraction analysis revealed the micro-domain structure of docetaxel in the polymeric microspheres. The spectrum showed a transformation of docetaxel from crystalline structure to amorphous microspheres. The average diameter of DOC-loaded microspheres was about 45 m. This, in combination with the surface morphology of microspheres suggested that the prepared docetaxel porous microspheres had uniform particle size, stable drug loading and sustained drug release behavior by the emulsification and solvent evaporation technique.
Compared with the rapid clearance of nanoparticles or micelles from peritoneal cavity, retention time of microspheres is much longer and peritoneal concentration is higher [40]. The much slower release of docetaxel from PLLA–L121–PLLA microspheres in comparison with free docetaxel can be attributed to the molecular structural characteristics of polymeric microspheres. This delay of drug release indicates their potential applicability in drug carrier to minimize the exposure of healthy tissues while increasing the accumulation of therapeutic drug in the tumor site. Moreover, cytotoxicity results revealed that the DOC-loaded microspheres remained the comparable anticancer effect even though docetaxel was released in an extended behavior. Pluronic® block copolymers were used in the study for its commercial availability, biocompatibility and safety [41]. Moreover, studies have reported that Pluronic block copolymers sensitized the resistant cancer cell lines, so that the cytotoxic activity of the drug could increase by 2–3 orders of magnitude [42]. The interstitial chemotherapy by local injection of sustainedrelease medication has many advantages, including minimal invasion, high drug concentration in the injection local, and lowered systemic toxicity, etc. The results of DOC-loaded microspheres chemotherapy showed significant anticancer effect on the mice bearing colorectal peritoneal carcinomatosis. It is considered that the increased survival rate of mice treated with DOC-loaded microspheres is probably due to the better antitumor efficacies of these particles. PLLA–L121–PLLA microspheres supplied active DOC molecules for extended times through a sustained release pattern, thereby exerting an antitumor effect in the CT26 colon carcinoma. Immunohistochemical staining with Ki-67 monoclonal antibody is a frequently used method for evaluation of cell proliferation. Sections of tumors from mice in each group were stained for Ki67 to determine the proliferation of tumor cells. According to Fig. 9A–D, weak Ki-67 immunoreactivity observed in tumor tissue from mice treated with DOC MS in comparison with free DOC, blank MS or NS. In Fig. 9E, Ki-67 LI of tumor tissues from DOC MS treated mice was significant lower compared with free DOC, blank MS, or
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Fig. 9. Ki-67 immunohistochemical staining of tumors. Representative Ki-67 immunohistochemical images of saline (A), blank MS (B), free DOC (C), and DOC MS (D) group, and mean Ki-67 LI in each group (E).
NS group. Results of Ki-67 staining of tumor tissues suggested that DOC MS could suppress tumor cell proliferation. The local injection of DOC-loaded microspheres demonstrated a significant antiangiogenic effect in our study. 5. Conclusion Biodegradable DOC MS were prepared and assigned for CRPC therapy. In vitro and in vivo toxicity evaluation indicated that after DOC was encapsulated into polymeric microspheres, in vitro cytotoxicity was increased compared with free DOC. Besides, a sustained in vitro release behavior was also observed in DOC MS group. Furthermore, compared with free DOC, intraperitoneal administration of DOC MS was more effective in suppressing tumor growth and metastasis and prolonged survival in vivo. Encapsulation of DOC in polymeric microspheres increased concentration and retention time in plasma and tumors. The PLLA–L121–PLLA block copolymers possess excellent biocompatibility because of the Pluronic and PLLA segments. In addition, the biodegradability of PLLA blocks makes it attractive for use in controlled drug release systems because the release rates can be controlled by varying the length and degradation rate of PLLA blocks. Due to its effectiveness and safety, the
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