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SN-38-Loaded PLGA microspheres injected intratumorally for cancer: preparation, characterization and evaluation
Journal of Drug Delivery Science and Technology 53 (2019) 101178
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Journal of Drug Delivery Science and Technology journal homepage: www.elsevier.com/locate/jddst
SN-38-Loaded PLGA microspheres injected intratumorally for cancer: preparation, characterization and evaluation
T
Yanyun Haoa, Jingjing Liua, Qingwen Jiab, Jiying Suic, Guihua Huanga,* a
Shandong University, No.44, Wenhua Xi Street, Ji'nan, 250012, China Shandong Academy of Pharmaceutical Sciences, No.989, Xinluo Street, Ji'nan, 250012, China c Affiliated Hospital of Shandong Academy of Medical Sciences No.38, Wuyingshan Street, Ji'nan, 250000, China b
ARTICLE INFO
ABSTRACT
Keywords: SN-38 PLGA Microspheres Intratumoral injection Cancer
7-ethyl-10-hydroxycamptothecin (SN-38), an active metabolite with broad spectrum cytotoxic activity produced by 7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxy camptothecin (CPT-11), has been shown to be useful in the treatment of various tumors. The aim of this research was to develop SN-38 loaded PLGA microspheres (SN-38-PLGA-MS) to extend retention time of SN-38 in tumor and reduce its concentrations in plasma, thereby to improve therapeutic efficacy and reduce side effects. The SN-38-PLGA-MS was prepared by emulsion-solvent evaporation method at a molar ratio of 1:10 (SN-38: PLGA). Tumor retention and plasma leakage were assessed by measuring drug concentrations in tumor sites and plasma at different time points. The therapeutic effect of SN-38-PLGA-MS was further evaluated by pharmacodynamic studies and histological analysis. The freeze-dried microspheres can effectively prolong the retention time of SN-38 in tumor site from 144 h to 432 h, which was 3.00 times than SN-38 solution. The in vivo pharmacodynamic results revealed that SN-38-PLGA-MS significantly improved the inhibition of tumor cell growth. In summary, SN-38-PLGA-MS was proved as a promising carrier for inhibiting tumor cell proliferation, and had important clinical value in the treatment of tumors.
1. Introduction It is well known that 7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxy camptothecin (CPT-11) [1], a semi-synthesized derivative of natural compound camptothecin (CPT), is a topoisomerase-I inhibitor with a broad spectrum cytotoxic activity against various tumors including colorectal, lung, liver and ovarian cancers [2,3]. Although CPT-11 has been clinically used in the form of hydrochloride injection, it still has some adverse reactions including diarrhea, vomiting, neutropenia, hair loss, shortness of breath, and fever at al [4,5]. In addition, there are two main transformation reactions of CPT-11, and the procedure is shown in Fig. 1. On the one hand, CPT-11 is a prodrug that can only exert anticancer effects after hydrolyzed into the active metabolite 7-ethyl-10-hydroxycamptothecin (SN-38) by various enzyme systems in the liver [6,7]. However, only 3%–4% of CPT-11 can be converted in humans. Furthermore, due to the equilibrium between its closed-loop and open-loop structure, only 55% of CPT-11 can keep intact closed-loop structure after injection [8]. On the other hand, for the SN-38 which has successfully been converted from CPT-11, another equilibrium between the active blocked-lactone ring and the inactive open-carboxylate structure appeared. Unfortunately, the conversed *
carboxylate structure tends to attach the albumin in plasma, motivates the equilibrium to shift right toward the conversion of more inactive open carboxylate structure [9]. Considering the low conversion rate of CPT-11 to SN-38 and the high conversion rate of blocked-lactone ring to open-carboxylate structure, in order to achieve the desirable therapeutic effects, a high and repeated dose of CPT-11 is required. However, this strategy not only suffers more severe side effects but also develops resistance against therapy. All the evidences mentioned above make CPT-11 no longer an excellent anti-tumor drug. Direct injection of SN-38 can achieve the same therapeutic effect at lower doses compared to CPT-11, indicating a greater dose advantage over CPT-11. However, the poor solubility of SN-38 in water and pharmaceutically acceptable organic solvents precludes its clinical application [10,11]. Additionally, the two modes of existence make the SN-38 unstable. Thirdly, SN-38 is a time-dependent drug, which means that increasing the residence time of the drug in the body may have a significant effect on improving the anti-tumor efficacy of SN-38. The physicochemical properties of SN-38 mentioned above are major obstacles to achieve effective treatment. Therefore, it is necessary to increase its solubility, stability and use an appropriate carrier to prolong its residence time in the body.
Corresponding author. Shandong University, 44 Wenhua Xi Street, Ji'nan, 250012, China. E-mail address: [email protected] (G. Huang).
https://doi.org/10.1016/j.jddst.2019.101178 Received 8 January 2019; Received in revised form 9 July 2019; Accepted 24 July 2019 Available online 25 July 2019 1773-2247/ © 2019 Elsevier B.V. All rights reserved.
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Fig. 1. The conversion process between CPT-11 and SN-38 and their respective open-loop and closed-loop structures.
2. Materials and methods
Microspheres have been increasingly studied for the controlled delivery of anticancer drugs [12,13]. Firstly, drugs dispersed on the surface or inside of the microspheres can be slowly released by a variety of mechanisms, including self-diffusion, transport, hydrolysis, and erosion, which are beneficial for increasing their retention in tumor [14–16]. Moreover, encapsulating the drugs into microspheres can improve their stabilities by protecting them from structural transformation or chemical degradation [17,18]. Finally, FDA-approved poly (lactic-co-glycolic-acid) (PLGA) becomes the most widely used material in microspheres for its good biocompatibility and biodegradability [19,20]. Acidic environment created by the degradation products of PLGA including poly lactic acid (PLA) and poly glycolic acid (PGA) will protect the lactone ring of SN-38 from hydrolysis, which improved the drug stability. Hence, PLGA microspheres could be an ideal drug-carrier for the clinical use of SN-38. However, due to the lactone ring of released SN-38 can strongly attach the albumin in plasma strongly [9], conventional drug administration may be not the best choice. Hence, localized delivery of SN-38 where released drug is limited to the tumor site is thought to improve its antitumor efficacy. In recent years, intratumoral injection of microsphere has attracted increasing attentions for its high drug concentration in local tumor and less leakage in plasma [21,22]. Benny et al. studied the antiangiogenic drugs encapsulated microspheres intratumorally for tumors, producing a good therapeutic effect by significantly reducing tumor volume and angiogenesis, thereby for the increased apoptosis [23]. A. Plotnikov demonstrated the advantage of intratumoral injection compared to intravenous administration under the same conditions also [24]. With the characters of simplified administration, improved efficacies and the decreased side effects, intratumoral injection possesses tremendous potential for clinical applications [25]. In this research, we developed SN-38 loaded PLGA microspheres (SN-38-PLGA-MS) by intratumorally injection for the treatment of cancer. Firstly, SN-38-PLGA-MS was prepared by emulsion solvent evaporation method [26–28] and the formulation of SN-38-PLGA-MS was optimized by orthogonal experiment. Next, the SN-38-PLGA-MS constructed above was evaluated by a series of characterizations including appearance and microscopic morphology, particle size, drug loading (DL), entrapment efficiency (EE) along with differential scanning calorimetry (DSC). Finally, in vivo kinetics and the anti-tumor efficiency of SN-38-PLGA-MS were confirmed by pharmacokinetic and pharmacodynamic experiments with H22-bearing Kunming mice.
2.1. Materials SN-38 was gained from Shanghai Longxiang Biomedical Development Co., Ltd. (Shanghai, China). PLGA (50:50, MW: 15,000) was obtained from Advanced Polymer Materials Inc. (Jinan, China). Polyvinyl alcohol (PVA, MW: 13,000–23,000; partial alcoholysis-typed 87–89) was purchased from Sigma Chemical Company. (USA). Dichloromethane was gained from Tianjin Guangcheng Reagent Co., Ltd. (Tianjin, China). Perchloric acid was provided by Tianjin Zheng Cheng Chemical Co., Ltd. (Tianjin, China). Dimethylsulfoxide was obtained from Tianjin Shield Specialty Chemical Co., Ltd. (Tianjin, China). All other chemical reagents used in this study were of analytical or chromatographical grade. 2.2. Preparation of SN-38-PLGA-MS SN-38-PLGA-MS was prepared by emulsion-solvent evaporation method [26–28]. Briefly, the accurately weighed mixture of SN-38 and PLGA (1:10) was dissolved in the mixture (dichlormethane: dimethyl sulfoxide = 4:1) to form the oil phase. The oil phase was scrolled fully and then pouring into 1.5 mL PVA aqueous solution (1.5%, W/V). After that, the mixture was sheared with a high-speed homogenizer of 5000 rpm for 1 min to form the O/W-typed primary emulsion. Subsequently, the O/W-typed emulsion was slowly added to 7.5 mL PVA aqueous solution (0.5%, W/V) under magnetic stirring at 400 rpm for 3 h to evaporate dichloromethane completely. SN-38-PLGA-MS was collected by high-speed centrifugation at 8000 rpm for 10 min to remove PVA residue. Finally, the suspension of SN-38-PLGA-MS was dispersed into water. 2.3. Optimization of formulation An L9 (34) orthogonal experiment [14,29] was selected to optimize the prescriptions and processes of SN-38-PLGA-MS on the basis of four factors. The factors included the PLGA concentration (A, mg/mL), the ratio of drug and carrier (B, W/W), the ratio of oil phase and aqueous phase (C, V/V), and the PVA concentration (D, %). The three levels of factor A were divided into 20, 30 and 40 mg/mL respectively. The three levels of factor B were divided into 1:5, 1:10 and 1:15 respectively. The three levels of factor C were divided into 1:1, 1:3 and 1:6 respectively. 2
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was added to dissolve the microspheres completely with the help of ultrasonic, then diluted the solution with the mixture to the mark. Remove 0.5 ml–5 ml volumetric flask and dilute to scale and detected in UV. Finally the EE and DL were calculated by the equations below [14,30] (1) and (2)
Table 1 Factor-level of L9 (34). Level
1 2 3
Factor A (mg/mL)
B(W/W)
C(V/V)
D (%)
20 30 40
1:5 1:10 1:15
1:1 1:3 1:6
0.5 1.0 1.5
EE(%) =
Wmeansure × 100% Wtotal
(1)
DL(%) =
Wmeasure × 100% WMS
(2)
The three levels of factor D were divided into 0.5, 1.0 and 1.5% respectively. Regarded DL and EE as the evaluation index for SN-38PLGA-MS, and then the best formulation was gained. The experimental design was showed in Table 1.
Where W total, W measure and WMS is the weight of SN-38 weighed accurately, the weight of SN-38 in the microspheres detected by UV and the weight of whole microsphere, respectively.
2.4. Freeze-dried powder of SN-38-PLGA-MS
2.6. Experimental animals
In order to improve the stability of the microspheres, we added lyoprotectant to form lyophilized powder. To screen the optimal amount of lyoprotectant, we first centrifuged the SN-38-PLGA-MS suspension to collect the precipitate. It was then washed 3 times. Finally, it was uniformly dispersed with distilled water, and then different concentrations of mannitol were added as the lyoprotectant. After that, microspheres powder was obtained by freeze-drying method. Specifically, well prepared suspension was pre-frozen in an ultra-cold freezer (MDF-382E, Sanyo Electric Co., Ltd., Japan) for 24 h and then transferred to the lyophilizer (FD5-2.5, GOLD SIM, Issaquah, WA) for 48 h. The effects of mannitol concentration on the appearance and redispersion time of microspheres were investigated, and the optimum mannitol concentration to prepare freeze-dried powder of SN-38-PLGAMS was screened out.
Female healthy Kunming mice (KM mice) ranged from 18 to 22 g were obtained from Medical Animal Test Center of Shandong University. All the experimental procedures were approved by the “Ethical Committee of Experimental Institutional Animal Center” of Shandong University (Approval Number: SCXY 20090001). All animal experiments comply with the ARRIVE guidelines and were carried out in accordance with the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978). 2.7. Pharmacokinetics studies 2.7.1. HPLC conditions The concentrations of SN-38 in plasma and tumor were determined by HPLC with a UV absorbance detector. The HPLC apparatus was a Shimadzu LC-10A system (Shimadzu, Kyoto, Japan) consisting of an LC10AT HPLC pump and an SPD-10A UV-VIS detector. Data processing was conducted by LC Solution software. Drug analysis was conducted on a PlatisilTM-ODS column (4.6 mm × 150 mm, Dikma Technologies, China). The mobile phase consisted of acetonitrile, pH 3.0 deionized water adjusted by phosphoric acid (35:65, v/v). Flow rate was 1.0 mL/ min and the injection volumes were 20 μL.
2.5. Characterization of SN-38-PLGA-MS 2.5.1. Appearance and morphological characterization of SN-38-PLGA-MS The morphology of freeze-dried SN-38-PLGA-MS powder was characterized firstly. Small amount of microsphere dispersed in deionized water was dropped onto a glass slide prepared in advance and then its morphology was observed by an LCD digital microscope (IDM2300, Jinan Yuhong Medical Equipment Co., Ltd). In order to further study the three-dimensional microscopic morphology of microspheres, the freeze-dried powder was also observed under the super depth microscope (SDM, Axio Scope A1 pol, Germany) and the scanning electron microscopy (SEM, JEOL 5800LV, Japan) respectively.
2.7.2. Sample preparation The collected blood samples were placed in the centrifuge tubes previously washed with heparin sodium. Then the blood samples were centrifuged at the speed of 3000 rpm for 10 min to collect plasma. At the same time, the dissected tumor tissues weighed in advance were placed in a 5 mL eppendorf tube, and 1 mL of physiological saline was added and homogenized using high speed homogenizer (DIAX900, Heidolph, Germany) to collect the tumor homogenate. After that, 200 μL collected plasma or tumor homogenate were added into eppendorf tubes and 200 μL acetonitrile and 200 μL 7% HClO4 were added, respectively, among which HClO4 was used to prevent the lactone form of SN-38 into carboxylate form as well as precipitate protein. Next, vortexed the mixture for 3 min, centrifuged for 10 min at 12000 rpm and filtered the supernatant layer through a 0.22 μm filter. Finally, injected 20 μL of the filtered supernatant layer into the HPLC to analyze the samples through the HPLC method mentioned above.
2.5.2. Particle size The particle size and its distribution of SN-38-PLGA-MS were measured by Mastersizer 2000 (Malvern Instruments Ltd). The freeze-dried SN-38-PLGA-MS powder was dispersed uniformly in distilled water and then measured at room temperature. The average value was gained and the size distributions were expressed in volume weighted mode. 2.5.3. Differential scanning calorimetry The thermal characteristics of the SN-38, PLGA, their physical mixture and the SN-38-PLGA-MS were measured by differential scanning calorimeter (DSC 204, Netzsch, Germany). The accurately weighed samples were sealed separately into a standard aluminum pan. DSC scanning was performed from 35–350 °C with heating rate of 10 °C/min under flowing nitrogen gas.
2.7.3. Design of pharmacokinetic study The study of in vivo pharmacokinetic was conducted by Sixty-six H22-bearing KM mice weighing 20 ± 2 g. They were divided into two groups randomly, one of which was given SN-38 solution via intratumoral injection at a concentration of 2 mg/mL, while the other was given SN-38-PLGA-MS (containing 2 mg/mL SN-38), the administration dosage of both was 10 mg/kg. After 0.5, 1, 4, 8, 12, 24, 48, 144, 240, 336, 432 h of administration respectively, 0.5 mL blood samples were taken out and the tumor tissues were stripped also.
2.5.4. Entrapment efficiency and drug loading The entrapment efficiency (EE) and drug loading (DL) of SN-38PLGA-MS was analyzed by ultraviolet spectrophotometer (UV–2102PCS, Shanghai Unico Instrument Company) [14]. Briefly, the SN-38-PLGA-MS prepared above was placed in 10 mL volumetric flask. 2 mL organic solvent mixture (dimethyl sulfoxide: acetonitrile = 1:1) 3
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2.8. Pharmacodynamics studies
Table 2 Results of orthogonal design experiments.
The study of in vivo pharmacokinetic was conducted on thirty-two H22 tumor-bearing KM mice weighing 20 ± 2 g. They were divided into four groups randomly. They were given the NS, blank PLGA microsphere, SN-38-Sol and SN-38-PLGA-MS via intratumoral injection at a concentration of 2 mg/mL, respectively. Each group was injected only once with the dose of 10 mg/kg. The body weight and tumor volume of mice in each group were weighed and measured on alternate days to ascertain possible systemic toxicity and evaluate antitumor efficacy of the two formulations. Tumor volume was calculated by the long sputum (L) and short diameter (W) of the tumor as eq follows (3):
Tumor volume =
L*W 2 2
No.
1 2 3 4 5 6 7 8 9 K1 K2 K3 R
(3)
To accurately assess the inhibitory effect of each group on tumor proliferation, we calculated tumor growth inhibition (TGI), tumor specific growth rate (SGR) and tumor doubling time (DT) [14,31]. TGI reflected the ability to inhibit tumor growth. SGR reflected the tumor growth rate. DT reflects the ability of drug to delay tumor growth. The value of those indicators can be calculated as the following Eq. (4) (5) (6), respectively.
TGI = SGR =
V1
V2 V1
× 100%
ln(V 3/ V 4) t
DT = ln2/SGR
Factors
EE (%)
A
B
C
D
1:5 1:10 1:15 1:5 1:10 1:15 1:5 1:10 1:15 67.703 64.640 67.900 3.260
80 80 80 120 120 120 160 160 160 53.233 78.607 68.403 25.374
1:1 1:3 1:6 1:3 1:6 1:1 1:6 1:1 1:3 76.680 54.223 69.340 22.457
0.5% 1.0% 1.5% 1.5% 0.5% 1.0% 1.0% 1.5% 0.5% 61.727 62.107 76.410 14.683
59.10 33.96 66.64 76.70 74.07 85.05 67.31 85.89 52.01
3 > 1 > 2; B: 2 > 3 > 1; C: 1 > 3 > 2; D: 3 > 2 > 1, so the best prescription combination was A3B2C1D3. That is to say, the composition of the prescription was determined as follows: the ratio of SN-38 and carrier (W/W) was 1:10, the PLGA concentrations were 120 mg/mL, the ratio of oil phase and aqueous phase was (V/V) is 1:1, and the PVA concentration was 1.5%. The preparation method was determined as follows: shear speed: 1 min; shear time: 5000 rpm.
(4) (5)
3.2. Freeze-dried powder of SN-38-PLGA-MS
(6)
The results of the effect of mannitol concentration on freeze-dried powder of SN-38-PLGA-MS were shown in Table 3. It can be seen from Fig. 2 and Table 3 that as the concentrations of mannitol increases, the appearance of SN-38-PLGA-MS lyophilized powder becomes more and more full, and the redissolution time of reconstitution is relatively shortened; However, when the amount of mannitol exceeds 5%, the redispersion time is prolonged. For a comprehensive analysis, 4% mannitol was selected as the lyoprotectant for SN-38-PLGA-MS.
Where V1 is the change of tumor volume in control group; V2 is the change of tumor volume in experimental group; V3 is the tumor volume after therapy; V4 is the tumor volume before therapy; T is the time interval between two measurements. 2.9. Histopathological analysis of tissue The histopathological analysis of tissue was carried out on sixty H22 bearing KM mice weighing 20 ± 2 g. They were divided into four groups randomly. The NS, blank PLGA microsphere, SN-38-Sol and SN38-PLGA-MS were given via intratumoral injection at a concentration of 2 mg/mL respectively. After 0, 1, 6, 12 and 18 days of administration respectively, the mice were sacrificed and the tumor tissues were stripped. Tumor tissue samples obtained were treated with formalin, paraffin embedded, and sectioned. Eighteen days later, all the mice were sacrificed. Then the tissues (tumor, heart, liver, spleen, lung, kidney and skin) were stripped and washed with NS. Then all the tissues sections were stained with hematoxylin and eosin for histopathological examination to evaluate in vivo antitumor efficacy and in vivo safety of all the groups.
3.3. Characterization of SN-38-PLGA-MS 3.3.1. Appearance and surface morphology The images of SN-38-PLGA-MS suspension prepared freshly (A), freeze-dried powder (B), and the reconstituted suspension of microspheres in deionized water (C) were shown in Fig. 3, which showed that the freeze-dried SN-38-PLGA-MS was white fluffy powder and can be uniformly dispersed. The surface morphology of SN-38-PLGA-MS under LCD digital microscope, SDM and SEM were shown in Fig. 4, which showed that the microspheres were smooth and well-dispersed.
2.10. Statistical analysis
3.3.2. Particle size As shown in supplementary information, the SN-38-PLGA-MS had an average size of (10.02 ± 0.05) μm, which was adequate for extending the half-life of SN-38, leading to a good sustained release in vivo.
All values were expressed as mean ± standard deviation (SD). The statistical analysis of significance between groups was assessed using unpaired Student's t-test with p < 0.05 indicating significant difference. An analysis of variance (ANOVA) test was also used if necessary. Pharmacokinetic parameters were obtained using DAS 2.0 (drug and statistics for windows) program.
Table 3 Effect of mannitol concentration on freeze-dried powder of SN-38-PLGA-MS.
3. Results and discussion 3.1. Orthogonal experiment As can be seen from Table 2, the order of influence of each factor on EE was as follows: B > C > D > A. K1, K2, and K3 refer to the total value of every level. The analysis results of the four factors were A: 4
Mannitol concentration (%)
Appearance Status
Redistribution Time (s)
1 2 3 4 5
White shrinkage powder White shrinkage powder White shrinkage powder White loose powder White loose powder, adherent
10 7 7 6 9
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Fig. 2. Photographs of lyophilized SN-38-PLGA-MS with different concentrations of mannitol protectants (1%, 2%, 3%, 4% and 5%, respectively). The top half of the picture was taken from the top view, the bottom half was taken from the front view, and the two are corresponded.
3.3.4. Determination of EE and DL According to the determination method in 2.5.4, the mean EE and DL of the SN-38-PLGA-MS were 81.91 ± 1.91 and 6.89 ± 0.07%, respectively. And there was no obvious difference in three batches of SN-38-PLGA-MS, showing a good process stability. In addition, the relatively high EE and DL can further improve the pharmacological effects on patients. 3.4. Pharmacokinetics studies 3.4.1. ConcentrationS of SN-38 in tumor site Mean concentrations of SN-38 versus time profiles in tumor after intratumoral injection of SN-38-Sol and SN-38-PLGA-MS were illustrated in Fig. 6 (A). The concentrations of SN-38 in solution group decreased from 160.86 to 57.48 μg/mL in 48 h and decreased to 3.94 μg/mL in 144 h, which indicated that lots of SN-38 were leaked from tumor. But, for SN-38-PLGA-MS group, the drug concentrations in 0.5 and 8 h were 236.49 and 134.96 μg/mL, which were 1.47-fold and 2.35-fold than SN-38-Sol, respectively. What is more, the drug concentrations of SN-38 decreased to 4.93 μg/mL in 432 h, which was much higher than that of SN-38-Sol in 144 h. Other than this, the concentrations of SN-38 in microsphere group at any time point were higher than that of SN-38-Sol, which indicated that the retention time of drug in tumor can be prolonged by encapsulating it into the microspheres.
Fig. 3. Photographs of lyophilized SN-38-PLGA-MS before lyophilization and after reconstitution: (A) and (a): Fresh-prepared SN-38-PLGA-MS. (B) and (b): Freeze-dried powder. (C) and (c): Reconstituted suspension of SN-38-PLGA-MS in deionized water. The top half of the picture was taken from the top view, the bottom half was taken from the front view, and the two are corresponded.
3.3.3. Differential scanning calorimetry The DSC curves of SN-38, blank PLGA-MS, their physical mixture, SN-38-PLGA-MS were displayed in Fig. 5. Three characteristic peaks at 158 °C, 235 °C and 286 °C was found in SN-38. For blank PLGA microsphere, a large typical absorption peak at 320 °C was showed. The peak of the mixture of SN-38 and blank PLGA microspheres was almost the superimposed peaks of SN-38 and blank PLGA microspheres. However, different from the peaks at around 158 °C, 235 °C and 286 °C (SN-38) and 320 °C (blank PLGA microsphere), a new absorption peak at 350 °C was showed in SN-38-PLGA-MS. The reasons can be explained as weakened crystallization degree of SN-38 when it encapsulated into microspheres, which can prevent the peaks of SN-38 crystals were detected. Comparing the absorption peaks in Fig. 5, we can conclude that SN-38-PLGA-MS has been synthesized successfully.
3.4.2. Results of pharmacokinetic parameters Main pharmacokinetic parameters of SN-38 in tumor after injection are shown in Table 4. The MRT of SN-38-PLGA-MS was 145.037 h, which was 4.60-fold than SN-38-Sol. In addition, the CL value of the SN-38-PLGA-MS was 0.22-fold than SN-38-Sol. Compared with the solution group, in microsphere group, the SN-38 retention time in tumor site was significantly extended. The AUC value of the SN-38-PLGA-MS was 3477.712 mg/L*h, which was 3.83-fold than SN-38-Sol, increasing the bioavailability of SN-38. Results analyzed above showed that after loading the SN-38 into microspheres, the SN-38 leakage rate from the tumor site to blood circulation was reduced, which can raise the biological utilization and reduce the toxic and side effect. 5
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Fig. 4. Microscopy photograph of SN-38-PLGA MS. (A) and (B) were the morphology under LCD digital microscope; (C) and (D) were the morphology under SDM; (E) and (F) were the morphology under SEM.
3.5. Pharmacodynamics studies 3.5.1. Change of tumor volume As can be seen from Fig. 7(A) and (B), after 18 days, the average tumor volumes of mice treated with NS and blank PLGA-MS reached about (2252.11 ± 393.22) mm3 and (2281.13 ± 860.76) mm3, respectively, and there were no significant differences in two groups (p > 0.05). The average tumor volumes of mice treated with SN-38-Sol and SN-38-PLGA MS were (1121.56 ± 562.72) mm3 and (605.59 ± 468.81) mm3, respectively. The results show that there were significant differences between SN-38-Sol group (p < 0.01) or SN-38-PLGA MS group (p < 0.001) and NS group. At the same time, we found that there was another significant difference in SN-38-Sol group and SN-38-PLGA MS group (p < 0.001). As can be seen from Fig. 7 (C), tumor weight shows the same tumor suppressing trend compared with tumor volume. As shown in Fig. 7 (D), SN-38-PLGA- MS group has the smallest tumor compared with other groups distinctly, showed a obvious tumor treatment effect. To further evaluate the advantages of microspheres in inhibiting cell proliferation, we calculated the values of TGI, DT, and SGR for each group. As shown in Table 5, the TGI, DT(d) and SGR (%/d) of SN-38PLGA-MS were 1.42 times, 1.97 times and 0.51 times than SN-38-Sol, respectively, showing a much stronger anti-tumor effect.
Fig. 5. The DSC curves of SN-38 (A), blank PLGA-MS (B), their physical mixture (C) and SN-38-PLGA-MS (D).
3.4.3. Concentrations of SN-38 in plasma Mean concentrations of SN-38 versus time profiles in plasma were illustrated in Fig. 6 (B). The drug concentrations in plasma of SN-38-Sol were obviously higher than SN-38-PLGA-MS. More specifically, the Cmax of SN-38-Sol group was 2.060 μg/mL, which is 3.07-fold than SN38-PLGA-MS group. Compared with the SN-38-Sol, encapsulating SN38 into microspheres can reduce the flow of the drug from local to blood due to the steric resistance contributed by suitable particle size. This result is consistent with the analysis above.
3.5.2. Change of body weight The body weight changes of the mice can be used to evaluate the systemic toxicity of different formations. As can be shown from Fig. 8 6
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Fig. 6. (A) Mean concentrations of SN-38 versus time profiles in tumor; (a) Amplified profiles of mean concentrations of SN-38 versus time in tumor on time ranged from 0 to 48 h; (B) Mean concentrations of SN-38 versus time profiles in plasma; (b) Amplified profiles of mean concentrations of SN-38 versus time in tumor on time ranged from 0 to 24 h. Data are expressed as mean ± SD, n = 3. Table 4 The main pharmacokinetic parameters of SN-38-Sol and SN-38-PLGA-MS after intratumoral injection (mean ± SD, n = 3). Analytical method
Parameters
SN-38-Sol
SN-38-PLGA-MS
Statistical moment
AUC0-t(mg/L*h) Cmax (mg/L) MRT CL(L/h/kg)
907.559 160.860 31.497 0.009
3477.712 236.490 145.037 0.002
Table 5 Results of TGI, DT and SGR. Group
TGI (%)
DT(d)
SGR(%/d)
NS group Blank MS group SN-38 solution group SN-38 MS group
– −7.03 60.90 86.57
7.10 6.88 11.92 23.45
9.76 10.08 5.82 2.96
increase speed in the latter was much faster than the former, which indicated that the SN-38-PLGA-MS had no obvious systemic toxicity. In addition, it can be analyzed from Fig. 8 (B) that an obvious difference in promoting mouse growth was found in SN-38-PLGA-MS group
(A), there were no significant differences between blank PLGA-MS group and the NS group. However, body weights of the mice in SN-38Sol group and SN-38-PLGA-MS group showed a stable increase, but the
Fig. 7. (A) The tumor volume versus time profiles; (B) Tumor volume of mice after 18 days of treatment in different preparation groups; (C) Tumor weight of mice after 18 days of treatment in different preparation groups. (D) Photograph of excised tumors, after treatment with NS, Blank MS, SN-38-Sol and SN-38-PLGA-MS. Data are expressed as mean ± SD, n = 8. Statistical significance was defined as **or ##p < 0.01 and ***p < 0.001. 7
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Fig. 8. (A) The body weight versus time profiles; Body weight of mice after 18 days of treatment NS, Blank MS, SN-38-Sol and SN-38-PLGA-MS. Data are expressed as mean ± SD, n = 8. Statistical significance was defined as **p < 0.01.
cannot inhibit tumor growth for a long time for its rapid release. However, in the SN-38-PLGA-MS group, most tumor cells were damaged with little ability of proliferation on the first day, but in the 6th day, severe necrosis was seen in central area of tumor cell the tumor cells with cell structure completely destroyed. And most tumor cells still on the 12th day were in necrosis as well as that of on the 6th day. But some of them began to proliferate on the 18th day. All the results obtained above indicated that the MS group has obvious sustained release effect, and can maintain the effective therapeutic concentration of the SN-38 at the tumor site for a long time comparing with the SN-38-Sol group. It can be concluded that SN-38-PLGA-MS had a stronger ability to suppress tumor growth than any other group. At the same time, the in vivo safety of the formulations administrated by intratumoral injection was evaluated by histopathological observation also. The H&E staining images of each tissue was shown in Fig. 10. There were no obvious differences in the cytoarchitecture of
compared with SN-38-Sol group (p < 0.01), which indicated SN-38PLGA-MS was a safe drug delivery system. 3.5.3. Results of histopathological analysis of tissues The light microscope images at 10X of tumor tissue after administration in different time of each group can be seen in Fig. 9. The dark purple area represents the proliferation of cells while the pink area means the cell has died or is dying. In the NS and the blank-PLGA-MS group, tumor cells were structurally intact and have strong proliferative capacity from the first day to the end, indicating that blank-PLGA-MS has no inhibitory effect on tumor growth. Whereas, in SN-38-Sol group, most of the tumor cells were damaged to broken flakes with lots of inflammatory cells infiltration on the first day. But on the 6th day, some tumors proliferated again. On the 18th day, the tumors proliferated faster, and it was no different from that without treatment, indicating that the SN-38-Sol
Fig. 9. H＆E stained images of tumors treated with NS, Blank PLGA MS, SN-38 Sol, SN-38-PLGA MS for tumor bearing mice at different times (10х). The scale bars were 200 μm. 8
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Fig. 10. H＆E stained images of main organs treated with NS, Blank PLGA MS, SN-38 Sol, SN-38-PLGA MS for tumor bearing mice at different times (10х). The scale bars were 200 μm.
microspheres should be carried out in a low temperature environment. In the part of pharmacokinetic studies, the normal KM mice were used as model animals. The concentrations determination of SN-38 in plasma and tumor samples was established by HPLC-UV method. After intratumoral injection, in the tumor, MRT and CL value for SN-38PLGA-MS group were 4.60 times and 0.22 times than the SN-38 solution group respectively, indicating that the retention time in tumor site of microspheres was extended significantly. While in the plasma, the Cmax in SN-38 solution group was 3.07 times than the SN-38-PLGA-MS group, which showed the higher leakage rate in the solution group. This is because molecules easily spread through the interstitial space after the intratumoral injection of the drug solution, so that the amount of the drug to maintain the local is small. Compared with the SN-38 solution, the release of SN-38 from microspheres is a slow process, which is helpful in increasing its tumor retention time. In vivo pharmacodynamic experiments were carried out using physiological saline as the negative control. The tumor volume of the NS group showed a rapid growth trend during the test period. 18-day test results showed that the mean tumor volume of MS group was the smallest. Subsequently, the tumor inhibition effect was quantitatively calculated. The results showed that the TGI, DT(d) and SGR (%/d) of SN-38-PLGA-MS were 1.42 times, 1.97 times and 0.51 times than those in SN-38-Sol, respectively, indicating that SN-38-PLGA-MS can better inhibit tumor growth compared with SN-38 solution. At the same time, compared with NS group, the mice body weight in SN-38-PLGA-MS
each tissue including heart, liver, spleen, lung, kidney and skin between the NS, blank-PLGA-MS, SN-38-Sol and the SN-38-PLGA-MS group. These results revealed that SN-38-PLGA-MS had good biocompatibility and low toxicity and was a promising candidate delivery carrier in our experimental conditions. 4. Discussion In this experiment, SN-38-PLGA-MS and free SN-38 were separated by ultra-low temperature centrifugation. Compared with the common methods such as dialysis, this method is simple to operate and has less drug leakage. Ultraviolet spectrophotometry was used to determine the drug content in SN-38-PLGA-MS. Because of the large amount of prescriptions, this method is simple, feasible and reliable. In this study, high-speed shearing under colostrum was carried out in the process of preparing SN-38-PLGA-MS, for the high-speed homogenizer could generate a large amount of heat at high speed, which had a certain influence on the stability of the system; If the temperature is much too high, the PLGA will become sticky and precipitate. In addition, low-temperature (4 °C) and high-speed centrifugation were also used in the centrifugal process of microspheres. It was found that if the SN-38-PLGA-MS suspension was centrifuged at the room temperature, the precipitated microspheres would turn into a pale yellow viscous rubbery aggregation and be difficult to be distracted in DMSO. In order to obtain SN-38-PLGA-MS, the steps of preparing and cleaning the 9
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group had increased, indicating that the SN-38-PLGA-MS prepared in this experiment had less toxic side effects. H&E slices of tumor tissue of mice showed that SN-38-PLGA-MS had a significant antitumor effect. H&E slices of the main organs showed that there were no significant differences between the SN-38-PLGA-MS group and the NS group, indicating that SN-38-PLGA-MS had no obvious side effects on the main organs of mice. All the results analyzed above showed that the SN-38-PLGA-MS prepared for introtumoral injection had certain clinical application potential.
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Siegel, Poly Lactic-co-Glycolic Acid (PLGA) as biodegradable controlled drug delivery carrier, Polymers 3 (3) (2011) 1377–1397 https://doi.org/ 10.3390/polym3031377. [21] J.B. Wolinsky, Y.L. Colson, M.W. Grinstaff, Local drug delivery strategies for cancer treatment: gels, nanoparticles, polymeric films, rods, and wafers, J. Control. Release 159 (1) (2012) 14–26 https://doi.org/10.1016/j.jconrel.2011.11.031. [22] J.J. Wei, X.M. Luo, M.H. Chen, J.F. Lu, X.H. Li, Spatial distribution and antitumor activities after intratumoral injection of fragmented fibers with loaded hydroxycamptothecin, Acta Biomater. 23 (2015) 189–200 https://doi.org/10.1016/j. actbio.2015.05.020. [23] O. Benny, S.K. Kim, K. Gvili, I.S. Radzishevsky, A. Mor, L. Verduzco, L.G. Menon, P.M. Black, M. Machluf, R.S. Carroll, In vivo fate and therapeutic efficacy of PF-4/ CTF microspheres in an orthotopic human glioblastoma model, FASEB J. 22 (2) (2008) 488–499 https://doi.org/10.1096/fj.07-8801com. [24] A. Plotnikov, D. Fishman, T. Tichler, R. Korenstein, Y. Keisari, Low electric field enhanced chemotherapy can cure mice with CT-26 colon carcinoma and induce anti-tumour immunity, Clin. Exp. Immunol. 138 (3) (2004) 410–416 https://doi. org/10.1111/j.1365-2249.2004.02636.x. [25] H.K.T. Liang, X.S. Lai, M.F. Wei, S.H. Lu, W.F. Wen, S.H. Kuo, C.M. Chen, W.Y.I. Tseng, F.H. Lin, Intratumoral injection of thermogelling and sustained-release carboplatin-loaded hydrogel simplifies the administration and remains the synergistic effect with radiotherapy for mice gliomas, Biomaterials 151 (2018) 38–52 https://doi.org/10.1016/j.biomaterials.2017.10.015. [26] L.B. Rodriguez, A. Avalos, N. Chiaia, A. Nadarajah, Effect of formulation and process parameters on chitosan microparticles prepared by an emulsion crosslinking technique, AAPS. PharmSciTech 18 (4) (2017) 1084–1094 https://doi.org/10. 1208/s12249-016-0677-x. [27] J.G. Feng, G.T. Yang, S.W. Zhang, Q. Liu, S.M. Jafari, D.J. 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5. Conclusion In this research, SN-38-PLGA-MS was prepared successfully by emulsion solvent evaporation method and showed several favorable performances as a drug delivery system including spherical particles, narrow size distribution, high entrapment efficiency and drug loading. In addition, pharmacokinetic results showed that compared with SN38-Sol group, SN-38-PLGA-MS can obviously extend the retention time of SN-38 in the tumor site and decreased its leakage into plasma, which significantly improved the treatment effect on tumor and reduce the side effects of drugs. What's more, the pharmacodynamics and histopathological analysis of tumors in different times also showed that the SN-38-PLGA-MS exhibited a higher antitumor efficacy than any other groups. All of these results indicate that intratumoral injection of SN-38PLGA-MS is a promising and effective strategy for cancer therapeutic, which makes it possible for the effective treatment of the patients and provides strong research support for its extensive clinical applications. Conflicts of interest None. Declaration of interest The authors declares that there are no conflict of interests in this work. Acknowledgment The authors extend sincere gratitude to the Shandong University for the experiment platform needed to carry out this work. The authors also thank Guihua Huang for her mentoring advice and he selfless help of the lab team members. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jddst.2019.101178. References [1] M.D. Alvau, S. Tartaggia, A. Meneghello, B. Casetta, G. Calia, P.A. Serra, F. Polo, G. Toffoli, Enzyme-based electrochemical biosensor for therapeutic drug monitoring of anticancer drug irinotecan, Anal. Chem. 90 (10) (2018) 6012–6019 https://doi.org/10.1021/acs.analchem.7b04357. [2] G. Vassal, M.J. Terrier-Lacombe, M.C. Bissery, A.M. Venuat, F. Gyergyay, J. Benard, J. Morizet, I. Boland, P. Ardouin, B. Bressac-De-Paillerets, A. Gouyette, Therapeutic activity of CPT-11, a DNA-topoisomerase I inhibitor, against peripheral primitive neuroectodermal tumour and neuroblastoma xenografts, Br. J. Canc. 74 (4) (1996) 537–545 https://doi.org/10.1208/s12249-019-1327-x. [3] X. Yang, Y. Yang, Q. Jia, Y. Hao, J. Liu, G. Huang, Preparation and evaluation of irinotecan Poly(Lactic-co-Glycolic Acid) nanoparticles for enhanced anti-tumor therapy, AAPS. PharmSciTech 20 (3) (2019) 133–134 https://doi.org/10.1038/bjc. 1996.398. [4] A. Stein, W. Voigt, K. Jordan, Chemotherapy-induced diarrhea: pathophysiology, frequency and guideline-based management, Ther. Adv. Med. Oncol. 2 (1) (2010) 51–63 https://doi.org/10.1177/1758834009355164.
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Y. Hao, et al. glycolide) microspheres and evaluation of pharmacokinetics and tissue distribution of BDMC-PLGA-MS in rats, Asian J. Pharm. Sci. 13 (1) (2018) 82–90 https://doi. org/10.1016/j.ajps.2017.09.002. [29] J. Emami, A. Pourmashhadi, H. Sadeghi, J. Varshosaz, H. Hamishehkar, Formulation and optimization of celecoxib-loaded PLGA nanoparticles by the Taguchi design and their in vitro cytotoxicity for lung cancer therapy, Pharm. Dev. Technol. 20 (7) (2015) 791–800 https://doi.org/10.3109/10837450.2014.920360.
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SN-38-Loaded PLGA microspheres injected intratumorally for cancer: preparation, characterization and evaluation
T
Yanyun Haoa, Jingjing Liua, Qingwen Jiab, Jiying Suic, Guihua Huanga,* a
Shandong University, No.44, Wenhua Xi Street, Ji'nan, 250012, China Shandong Academy of Pharmaceutical Sciences, No.989, Xinluo Street, Ji'nan, 250012, China c Affiliated Hospital of Shandong Academy of Medical Sciences No.38, Wuyingshan Street, Ji'nan, 250000, China b
ARTICLE INFO
ABSTRACT
Keywords: SN-38 PLGA Microspheres Intratumoral injection Cancer
7-ethyl-10-hydroxycamptothecin (SN-38), an active metabolite with broad spectrum cytotoxic activity produced by 7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxy camptothecin (CPT-11), has been shown to be useful in the treatment of various tumors. The aim of this research was to develop SN-38 loaded PLGA microspheres (SN-38-PLGA-MS) to extend retention time of SN-38 in tumor and reduce its concentrations in plasma, thereby to improve therapeutic efficacy and reduce side effects. The SN-38-PLGA-MS was prepared by emulsion-solvent evaporation method at a molar ratio of 1:10 (SN-38: PLGA). Tumor retention and plasma leakage were assessed by measuring drug concentrations in tumor sites and plasma at different time points. The therapeutic effect of SN-38-PLGA-MS was further evaluated by pharmacodynamic studies and histological analysis. The freeze-dried microspheres can effectively prolong the retention time of SN-38 in tumor site from 144 h to 432 h, which was 3.00 times than SN-38 solution. The in vivo pharmacodynamic results revealed that SN-38-PLGA-MS significantly improved the inhibition of tumor cell growth. In summary, SN-38-PLGA-MS was proved as a promising carrier for inhibiting tumor cell proliferation, and had important clinical value in the treatment of tumors.
1. Introduction It is well known that 7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxy camptothecin (CPT-11) [1], a semi-synthesized derivative of natural compound camptothecin (CPT), is a topoisomerase-I inhibitor with a broad spectrum cytotoxic activity against various tumors including colorectal, lung, liver and ovarian cancers [2,3]. Although CPT-11 has been clinically used in the form of hydrochloride injection, it still has some adverse reactions including diarrhea, vomiting, neutropenia, hair loss, shortness of breath, and fever at al [4,5]. In addition, there are two main transformation reactions of CPT-11, and the procedure is shown in Fig. 1. On the one hand, CPT-11 is a prodrug that can only exert anticancer effects after hydrolyzed into the active metabolite 7-ethyl-10-hydroxycamptothecin (SN-38) by various enzyme systems in the liver [6,7]. However, only 3%–4% of CPT-11 can be converted in humans. Furthermore, due to the equilibrium between its closed-loop and open-loop structure, only 55% of CPT-11 can keep intact closed-loop structure after injection [8]. On the other hand, for the SN-38 which has successfully been converted from CPT-11, another equilibrium between the active blocked-lactone ring and the inactive open-carboxylate structure appeared. Unfortunately, the conversed *
carboxylate structure tends to attach the albumin in plasma, motivates the equilibrium to shift right toward the conversion of more inactive open carboxylate structure [9]. Considering the low conversion rate of CPT-11 to SN-38 and the high conversion rate of blocked-lactone ring to open-carboxylate structure, in order to achieve the desirable therapeutic effects, a high and repeated dose of CPT-11 is required. However, this strategy not only suffers more severe side effects but also develops resistance against therapy. All the evidences mentioned above make CPT-11 no longer an excellent anti-tumor drug. Direct injection of SN-38 can achieve the same therapeutic effect at lower doses compared to CPT-11, indicating a greater dose advantage over CPT-11. However, the poor solubility of SN-38 in water and pharmaceutically acceptable organic solvents precludes its clinical application [10,11]. Additionally, the two modes of existence make the SN-38 unstable. Thirdly, SN-38 is a time-dependent drug, which means that increasing the residence time of the drug in the body may have a significant effect on improving the anti-tumor efficacy of SN-38. The physicochemical properties of SN-38 mentioned above are major obstacles to achieve effective treatment. Therefore, it is necessary to increase its solubility, stability and use an appropriate carrier to prolong its residence time in the body.
Corresponding author. Shandong University, 44 Wenhua Xi Street, Ji'nan, 250012, China. E-mail address: [email protected] (G. Huang).
https://doi.org/10.1016/j.jddst.2019.101178 Received 8 January 2019; Received in revised form 9 July 2019; Accepted 24 July 2019 Available online 25 July 2019 1773-2247/ © 2019 Elsevier B.V. All rights reserved.
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Fig. 1. The conversion process between CPT-11 and SN-38 and their respective open-loop and closed-loop structures.
2. Materials and methods
Microspheres have been increasingly studied for the controlled delivery of anticancer drugs [12,13]. Firstly, drugs dispersed on the surface or inside of the microspheres can be slowly released by a variety of mechanisms, including self-diffusion, transport, hydrolysis, and erosion, which are beneficial for increasing their retention in tumor [14–16]. Moreover, encapsulating the drugs into microspheres can improve their stabilities by protecting them from structural transformation or chemical degradation [17,18]. Finally, FDA-approved poly (lactic-co-glycolic-acid) (PLGA) becomes the most widely used material in microspheres for its good biocompatibility and biodegradability [19,20]. Acidic environment created by the degradation products of PLGA including poly lactic acid (PLA) and poly glycolic acid (PGA) will protect the lactone ring of SN-38 from hydrolysis, which improved the drug stability. Hence, PLGA microspheres could be an ideal drug-carrier for the clinical use of SN-38. However, due to the lactone ring of released SN-38 can strongly attach the albumin in plasma strongly [9], conventional drug administration may be not the best choice. Hence, localized delivery of SN-38 where released drug is limited to the tumor site is thought to improve its antitumor efficacy. In recent years, intratumoral injection of microsphere has attracted increasing attentions for its high drug concentration in local tumor and less leakage in plasma [21,22]. Benny et al. studied the antiangiogenic drugs encapsulated microspheres intratumorally for tumors, producing a good therapeutic effect by significantly reducing tumor volume and angiogenesis, thereby for the increased apoptosis [23]. A. Plotnikov demonstrated the advantage of intratumoral injection compared to intravenous administration under the same conditions also [24]. With the characters of simplified administration, improved efficacies and the decreased side effects, intratumoral injection possesses tremendous potential for clinical applications [25]. In this research, we developed SN-38 loaded PLGA microspheres (SN-38-PLGA-MS) by intratumorally injection for the treatment of cancer. Firstly, SN-38-PLGA-MS was prepared by emulsion solvent evaporation method [26–28] and the formulation of SN-38-PLGA-MS was optimized by orthogonal experiment. Next, the SN-38-PLGA-MS constructed above was evaluated by a series of characterizations including appearance and microscopic morphology, particle size, drug loading (DL), entrapment efficiency (EE) along with differential scanning calorimetry (DSC). Finally, in vivo kinetics and the anti-tumor efficiency of SN-38-PLGA-MS were confirmed by pharmacokinetic and pharmacodynamic experiments with H22-bearing Kunming mice.
2.1. Materials SN-38 was gained from Shanghai Longxiang Biomedical Development Co., Ltd. (Shanghai, China). PLGA (50:50, MW: 15,000) was obtained from Advanced Polymer Materials Inc. (Jinan, China). Polyvinyl alcohol (PVA, MW: 13,000–23,000; partial alcoholysis-typed 87–89) was purchased from Sigma Chemical Company. (USA). Dichloromethane was gained from Tianjin Guangcheng Reagent Co., Ltd. (Tianjin, China). Perchloric acid was provided by Tianjin Zheng Cheng Chemical Co., Ltd. (Tianjin, China). Dimethylsulfoxide was obtained from Tianjin Shield Specialty Chemical Co., Ltd. (Tianjin, China). All other chemical reagents used in this study were of analytical or chromatographical grade. 2.2. Preparation of SN-38-PLGA-MS SN-38-PLGA-MS was prepared by emulsion-solvent evaporation method [26–28]. Briefly, the accurately weighed mixture of SN-38 and PLGA (1:10) was dissolved in the mixture (dichlormethane: dimethyl sulfoxide = 4:1) to form the oil phase. The oil phase was scrolled fully and then pouring into 1.5 mL PVA aqueous solution (1.5%, W/V). After that, the mixture was sheared with a high-speed homogenizer of 5000 rpm for 1 min to form the O/W-typed primary emulsion. Subsequently, the O/W-typed emulsion was slowly added to 7.5 mL PVA aqueous solution (0.5%, W/V) under magnetic stirring at 400 rpm for 3 h to evaporate dichloromethane completely. SN-38-PLGA-MS was collected by high-speed centrifugation at 8000 rpm for 10 min to remove PVA residue. Finally, the suspension of SN-38-PLGA-MS was dispersed into water. 2.3. Optimization of formulation An L9 (34) orthogonal experiment [14,29] was selected to optimize the prescriptions and processes of SN-38-PLGA-MS on the basis of four factors. The factors included the PLGA concentration (A, mg/mL), the ratio of drug and carrier (B, W/W), the ratio of oil phase and aqueous phase (C, V/V), and the PVA concentration (D, %). The three levels of factor A were divided into 20, 30 and 40 mg/mL respectively. The three levels of factor B were divided into 1:5, 1:10 and 1:15 respectively. The three levels of factor C were divided into 1:1, 1:3 and 1:6 respectively. 2
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was added to dissolve the microspheres completely with the help of ultrasonic, then diluted the solution with the mixture to the mark. Remove 0.5 ml–5 ml volumetric flask and dilute to scale and detected in UV. Finally the EE and DL were calculated by the equations below [14,30] (1) and (2)
Table 1 Factor-level of L9 (34). Level
1 2 3
Factor A (mg/mL)
B(W/W)
C(V/V)
D (%)
20 30 40
1:5 1:10 1:15
1:1 1:3 1:6
0.5 1.0 1.5
EE(%) =
Wmeansure × 100% Wtotal
(1)
DL(%) =
Wmeasure × 100% WMS
(2)
The three levels of factor D were divided into 0.5, 1.0 and 1.5% respectively. Regarded DL and EE as the evaluation index for SN-38PLGA-MS, and then the best formulation was gained. The experimental design was showed in Table 1.
Where W total, W measure and WMS is the weight of SN-38 weighed accurately, the weight of SN-38 in the microspheres detected by UV and the weight of whole microsphere, respectively.
2.4. Freeze-dried powder of SN-38-PLGA-MS
2.6. Experimental animals
In order to improve the stability of the microspheres, we added lyoprotectant to form lyophilized powder. To screen the optimal amount of lyoprotectant, we first centrifuged the SN-38-PLGA-MS suspension to collect the precipitate. It was then washed 3 times. Finally, it was uniformly dispersed with distilled water, and then different concentrations of mannitol were added as the lyoprotectant. After that, microspheres powder was obtained by freeze-drying method. Specifically, well prepared suspension was pre-frozen in an ultra-cold freezer (MDF-382E, Sanyo Electric Co., Ltd., Japan) for 24 h and then transferred to the lyophilizer (FD5-2.5, GOLD SIM, Issaquah, WA) for 48 h. The effects of mannitol concentration on the appearance and redispersion time of microspheres were investigated, and the optimum mannitol concentration to prepare freeze-dried powder of SN-38-PLGAMS was screened out.
Female healthy Kunming mice (KM mice) ranged from 18 to 22 g were obtained from Medical Animal Test Center of Shandong University. All the experimental procedures were approved by the “Ethical Committee of Experimental Institutional Animal Center” of Shandong University (Approval Number: SCXY 20090001). All animal experiments comply with the ARRIVE guidelines and were carried out in accordance with the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978). 2.7. Pharmacokinetics studies 2.7.1. HPLC conditions The concentrations of SN-38 in plasma and tumor were determined by HPLC with a UV absorbance detector. The HPLC apparatus was a Shimadzu LC-10A system (Shimadzu, Kyoto, Japan) consisting of an LC10AT HPLC pump and an SPD-10A UV-VIS detector. Data processing was conducted by LC Solution software. Drug analysis was conducted on a PlatisilTM-ODS column (4.6 mm × 150 mm, Dikma Technologies, China). The mobile phase consisted of acetonitrile, pH 3.0 deionized water adjusted by phosphoric acid (35:65, v/v). Flow rate was 1.0 mL/ min and the injection volumes were 20 μL.
2.5. Characterization of SN-38-PLGA-MS 2.5.1. Appearance and morphological characterization of SN-38-PLGA-MS The morphology of freeze-dried SN-38-PLGA-MS powder was characterized firstly. Small amount of microsphere dispersed in deionized water was dropped onto a glass slide prepared in advance and then its morphology was observed by an LCD digital microscope (IDM2300, Jinan Yuhong Medical Equipment Co., Ltd). In order to further study the three-dimensional microscopic morphology of microspheres, the freeze-dried powder was also observed under the super depth microscope (SDM, Axio Scope A1 pol, Germany) and the scanning electron microscopy (SEM, JEOL 5800LV, Japan) respectively.
2.7.2. Sample preparation The collected blood samples were placed in the centrifuge tubes previously washed with heparin sodium. Then the blood samples were centrifuged at the speed of 3000 rpm for 10 min to collect plasma. At the same time, the dissected tumor tissues weighed in advance were placed in a 5 mL eppendorf tube, and 1 mL of physiological saline was added and homogenized using high speed homogenizer (DIAX900, Heidolph, Germany) to collect the tumor homogenate. After that, 200 μL collected plasma or tumor homogenate were added into eppendorf tubes and 200 μL acetonitrile and 200 μL 7% HClO4 were added, respectively, among which HClO4 was used to prevent the lactone form of SN-38 into carboxylate form as well as precipitate protein. Next, vortexed the mixture for 3 min, centrifuged for 10 min at 12000 rpm and filtered the supernatant layer through a 0.22 μm filter. Finally, injected 20 μL of the filtered supernatant layer into the HPLC to analyze the samples through the HPLC method mentioned above.
2.5.2. Particle size The particle size and its distribution of SN-38-PLGA-MS were measured by Mastersizer 2000 (Malvern Instruments Ltd). The freeze-dried SN-38-PLGA-MS powder was dispersed uniformly in distilled water and then measured at room temperature. The average value was gained and the size distributions were expressed in volume weighted mode. 2.5.3. Differential scanning calorimetry The thermal characteristics of the SN-38, PLGA, their physical mixture and the SN-38-PLGA-MS were measured by differential scanning calorimeter (DSC 204, Netzsch, Germany). The accurately weighed samples were sealed separately into a standard aluminum pan. DSC scanning was performed from 35–350 °C with heating rate of 10 °C/min under flowing nitrogen gas.
2.7.3. Design of pharmacokinetic study The study of in vivo pharmacokinetic was conducted by Sixty-six H22-bearing KM mice weighing 20 ± 2 g. They were divided into two groups randomly, one of which was given SN-38 solution via intratumoral injection at a concentration of 2 mg/mL, while the other was given SN-38-PLGA-MS (containing 2 mg/mL SN-38), the administration dosage of both was 10 mg/kg. After 0.5, 1, 4, 8, 12, 24, 48, 144, 240, 336, 432 h of administration respectively, 0.5 mL blood samples were taken out and the tumor tissues were stripped also.
2.5.4. Entrapment efficiency and drug loading The entrapment efficiency (EE) and drug loading (DL) of SN-38PLGA-MS was analyzed by ultraviolet spectrophotometer (UV–2102PCS, Shanghai Unico Instrument Company) [14]. Briefly, the SN-38-PLGA-MS prepared above was placed in 10 mL volumetric flask. 2 mL organic solvent mixture (dimethyl sulfoxide: acetonitrile = 1:1) 3
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2.8. Pharmacodynamics studies
Table 2 Results of orthogonal design experiments.
The study of in vivo pharmacokinetic was conducted on thirty-two H22 tumor-bearing KM mice weighing 20 ± 2 g. They were divided into four groups randomly. They were given the NS, blank PLGA microsphere, SN-38-Sol and SN-38-PLGA-MS via intratumoral injection at a concentration of 2 mg/mL, respectively. Each group was injected only once with the dose of 10 mg/kg. The body weight and tumor volume of mice in each group were weighed and measured on alternate days to ascertain possible systemic toxicity and evaluate antitumor efficacy of the two formulations. Tumor volume was calculated by the long sputum (L) and short diameter (W) of the tumor as eq follows (3):
Tumor volume =
L*W 2 2
No.
1 2 3 4 5 6 7 8 9 K1 K2 K3 R
(3)
To accurately assess the inhibitory effect of each group on tumor proliferation, we calculated tumor growth inhibition (TGI), tumor specific growth rate (SGR) and tumor doubling time (DT) [14,31]. TGI reflected the ability to inhibit tumor growth. SGR reflected the tumor growth rate. DT reflects the ability of drug to delay tumor growth. The value of those indicators can be calculated as the following Eq. (4) (5) (6), respectively.
TGI = SGR =
V1
V2 V1
× 100%
ln(V 3/ V 4) t
DT = ln2/SGR
Factors
EE (%)
A
B
C
D
1:5 1:10 1:15 1:5 1:10 1:15 1:5 1:10 1:15 67.703 64.640 67.900 3.260
80 80 80 120 120 120 160 160 160 53.233 78.607 68.403 25.374
1:1 1:3 1:6 1:3 1:6 1:1 1:6 1:1 1:3 76.680 54.223 69.340 22.457
0.5% 1.0% 1.5% 1.5% 0.5% 1.0% 1.0% 1.5% 0.5% 61.727 62.107 76.410 14.683
59.10 33.96 66.64 76.70 74.07 85.05 67.31 85.89 52.01
3 > 1 > 2; B: 2 > 3 > 1; C: 1 > 3 > 2; D: 3 > 2 > 1, so the best prescription combination was A3B2C1D3. That is to say, the composition of the prescription was determined as follows: the ratio of SN-38 and carrier (W/W) was 1:10, the PLGA concentrations were 120 mg/mL, the ratio of oil phase and aqueous phase was (V/V) is 1:1, and the PVA concentration was 1.5%. The preparation method was determined as follows: shear speed: 1 min; shear time: 5000 rpm.
(4) (5)
3.2. Freeze-dried powder of SN-38-PLGA-MS
(6)
The results of the effect of mannitol concentration on freeze-dried powder of SN-38-PLGA-MS were shown in Table 3. It can be seen from Fig. 2 and Table 3 that as the concentrations of mannitol increases, the appearance of SN-38-PLGA-MS lyophilized powder becomes more and more full, and the redissolution time of reconstitution is relatively shortened; However, when the amount of mannitol exceeds 5%, the redispersion time is prolonged. For a comprehensive analysis, 4% mannitol was selected as the lyoprotectant for SN-38-PLGA-MS.
Where V1 is the change of tumor volume in control group; V2 is the change of tumor volume in experimental group; V3 is the tumor volume after therapy; V4 is the tumor volume before therapy; T is the time interval between two measurements. 2.9. Histopathological analysis of tissue The histopathological analysis of tissue was carried out on sixty H22 bearing KM mice weighing 20 ± 2 g. They were divided into four groups randomly. The NS, blank PLGA microsphere, SN-38-Sol and SN38-PLGA-MS were given via intratumoral injection at a concentration of 2 mg/mL respectively. After 0, 1, 6, 12 and 18 days of administration respectively, the mice were sacrificed and the tumor tissues were stripped. Tumor tissue samples obtained were treated with formalin, paraffin embedded, and sectioned. Eighteen days later, all the mice were sacrificed. Then the tissues (tumor, heart, liver, spleen, lung, kidney and skin) were stripped and washed with NS. Then all the tissues sections were stained with hematoxylin and eosin for histopathological examination to evaluate in vivo antitumor efficacy and in vivo safety of all the groups.
3.3. Characterization of SN-38-PLGA-MS 3.3.1. Appearance and surface morphology The images of SN-38-PLGA-MS suspension prepared freshly (A), freeze-dried powder (B), and the reconstituted suspension of microspheres in deionized water (C) were shown in Fig. 3, which showed that the freeze-dried SN-38-PLGA-MS was white fluffy powder and can be uniformly dispersed. The surface morphology of SN-38-PLGA-MS under LCD digital microscope, SDM and SEM were shown in Fig. 4, which showed that the microspheres were smooth and well-dispersed.
2.10. Statistical analysis
3.3.2. Particle size As shown in supplementary information, the SN-38-PLGA-MS had an average size of (10.02 ± 0.05) μm, which was adequate for extending the half-life of SN-38, leading to a good sustained release in vivo.
All values were expressed as mean ± standard deviation (SD). The statistical analysis of significance between groups was assessed using unpaired Student's t-test with p < 0.05 indicating significant difference. An analysis of variance (ANOVA) test was also used if necessary. Pharmacokinetic parameters were obtained using DAS 2.0 (drug and statistics for windows) program.
Table 3 Effect of mannitol concentration on freeze-dried powder of SN-38-PLGA-MS.
3. Results and discussion 3.1. Orthogonal experiment As can be seen from Table 2, the order of influence of each factor on EE was as follows: B > C > D > A. K1, K2, and K3 refer to the total value of every level. The analysis results of the four factors were A: 4
Mannitol concentration (%)
Appearance Status
Redistribution Time (s)
1 2 3 4 5
White shrinkage powder White shrinkage powder White shrinkage powder White loose powder White loose powder, adherent
10 7 7 6 9
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Fig. 2. Photographs of lyophilized SN-38-PLGA-MS with different concentrations of mannitol protectants (1%, 2%, 3%, 4% and 5%, respectively). The top half of the picture was taken from the top view, the bottom half was taken from the front view, and the two are corresponded.
3.3.4. Determination of EE and DL According to the determination method in 2.5.4, the mean EE and DL of the SN-38-PLGA-MS were 81.91 ± 1.91 and 6.89 ± 0.07%, respectively. And there was no obvious difference in three batches of SN-38-PLGA-MS, showing a good process stability. In addition, the relatively high EE and DL can further improve the pharmacological effects on patients. 3.4. Pharmacokinetics studies 3.4.1. ConcentrationS of SN-38 in tumor site Mean concentrations of SN-38 versus time profiles in tumor after intratumoral injection of SN-38-Sol and SN-38-PLGA-MS were illustrated in Fig. 6 (A). The concentrations of SN-38 in solution group decreased from 160.86 to 57.48 μg/mL in 48 h and decreased to 3.94 μg/mL in 144 h, which indicated that lots of SN-38 were leaked from tumor. But, for SN-38-PLGA-MS group, the drug concentrations in 0.5 and 8 h were 236.49 and 134.96 μg/mL, which were 1.47-fold and 2.35-fold than SN-38-Sol, respectively. What is more, the drug concentrations of SN-38 decreased to 4.93 μg/mL in 432 h, which was much higher than that of SN-38-Sol in 144 h. Other than this, the concentrations of SN-38 in microsphere group at any time point were higher than that of SN-38-Sol, which indicated that the retention time of drug in tumor can be prolonged by encapsulating it into the microspheres.
Fig. 3. Photographs of lyophilized SN-38-PLGA-MS before lyophilization and after reconstitution: (A) and (a): Fresh-prepared SN-38-PLGA-MS. (B) and (b): Freeze-dried powder. (C) and (c): Reconstituted suspension of SN-38-PLGA-MS in deionized water. The top half of the picture was taken from the top view, the bottom half was taken from the front view, and the two are corresponded.
3.3.3. Differential scanning calorimetry The DSC curves of SN-38, blank PLGA-MS, their physical mixture, SN-38-PLGA-MS were displayed in Fig. 5. Three characteristic peaks at 158 °C, 235 °C and 286 °C was found in SN-38. For blank PLGA microsphere, a large typical absorption peak at 320 °C was showed. The peak of the mixture of SN-38 and blank PLGA microspheres was almost the superimposed peaks of SN-38 and blank PLGA microspheres. However, different from the peaks at around 158 °C, 235 °C and 286 °C (SN-38) and 320 °C (blank PLGA microsphere), a new absorption peak at 350 °C was showed in SN-38-PLGA-MS. The reasons can be explained as weakened crystallization degree of SN-38 when it encapsulated into microspheres, which can prevent the peaks of SN-38 crystals were detected. Comparing the absorption peaks in Fig. 5, we can conclude that SN-38-PLGA-MS has been synthesized successfully.
3.4.2. Results of pharmacokinetic parameters Main pharmacokinetic parameters of SN-38 in tumor after injection are shown in Table 4. The MRT of SN-38-PLGA-MS was 145.037 h, which was 4.60-fold than SN-38-Sol. In addition, the CL value of the SN-38-PLGA-MS was 0.22-fold than SN-38-Sol. Compared with the solution group, in microsphere group, the SN-38 retention time in tumor site was significantly extended. The AUC value of the SN-38-PLGA-MS was 3477.712 mg/L*h, which was 3.83-fold than SN-38-Sol, increasing the bioavailability of SN-38. Results analyzed above showed that after loading the SN-38 into microspheres, the SN-38 leakage rate from the tumor site to blood circulation was reduced, which can raise the biological utilization and reduce the toxic and side effect. 5
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Fig. 4. Microscopy photograph of SN-38-PLGA MS. (A) and (B) were the morphology under LCD digital microscope; (C) and (D) were the morphology under SDM; (E) and (F) were the morphology under SEM.
3.5. Pharmacodynamics studies 3.5.1. Change of tumor volume As can be seen from Fig. 7(A) and (B), after 18 days, the average tumor volumes of mice treated with NS and blank PLGA-MS reached about (2252.11 ± 393.22) mm3 and (2281.13 ± 860.76) mm3, respectively, and there were no significant differences in two groups (p > 0.05). The average tumor volumes of mice treated with SN-38-Sol and SN-38-PLGA MS were (1121.56 ± 562.72) mm3 and (605.59 ± 468.81) mm3, respectively. The results show that there were significant differences between SN-38-Sol group (p < 0.01) or SN-38-PLGA MS group (p < 0.001) and NS group. At the same time, we found that there was another significant difference in SN-38-Sol group and SN-38-PLGA MS group (p < 0.001). As can be seen from Fig. 7 (C), tumor weight shows the same tumor suppressing trend compared with tumor volume. As shown in Fig. 7 (D), SN-38-PLGA- MS group has the smallest tumor compared with other groups distinctly, showed a obvious tumor treatment effect. To further evaluate the advantages of microspheres in inhibiting cell proliferation, we calculated the values of TGI, DT, and SGR for each group. As shown in Table 5, the TGI, DT(d) and SGR (%/d) of SN-38PLGA-MS were 1.42 times, 1.97 times and 0.51 times than SN-38-Sol, respectively, showing a much stronger anti-tumor effect.
Fig. 5. The DSC curves of SN-38 (A), blank PLGA-MS (B), their physical mixture (C) and SN-38-PLGA-MS (D).
3.4.3. Concentrations of SN-38 in plasma Mean concentrations of SN-38 versus time profiles in plasma were illustrated in Fig. 6 (B). The drug concentrations in plasma of SN-38-Sol were obviously higher than SN-38-PLGA-MS. More specifically, the Cmax of SN-38-Sol group was 2.060 μg/mL, which is 3.07-fold than SN38-PLGA-MS group. Compared with the SN-38-Sol, encapsulating SN38 into microspheres can reduce the flow of the drug from local to blood due to the steric resistance contributed by suitable particle size. This result is consistent with the analysis above.
3.5.2. Change of body weight The body weight changes of the mice can be used to evaluate the systemic toxicity of different formations. As can be shown from Fig. 8 6
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Fig. 6. (A) Mean concentrations of SN-38 versus time profiles in tumor; (a) Amplified profiles of mean concentrations of SN-38 versus time in tumor on time ranged from 0 to 48 h; (B) Mean concentrations of SN-38 versus time profiles in plasma; (b) Amplified profiles of mean concentrations of SN-38 versus time in tumor on time ranged from 0 to 24 h. Data are expressed as mean ± SD, n = 3. Table 4 The main pharmacokinetic parameters of SN-38-Sol and SN-38-PLGA-MS after intratumoral injection (mean ± SD, n = 3). Analytical method
Parameters
SN-38-Sol
SN-38-PLGA-MS
Statistical moment
AUC0-t(mg/L*h) Cmax (mg/L) MRT CL(L/h/kg)
907.559 160.860 31.497 0.009
3477.712 236.490 145.037 0.002
Table 5 Results of TGI, DT and SGR. Group
TGI (%)
DT(d)
SGR(%/d)
NS group Blank MS group SN-38 solution group SN-38 MS group
– −7.03 60.90 86.57
7.10 6.88 11.92 23.45
9.76 10.08 5.82 2.96
increase speed in the latter was much faster than the former, which indicated that the SN-38-PLGA-MS had no obvious systemic toxicity. In addition, it can be analyzed from Fig. 8 (B) that an obvious difference in promoting mouse growth was found in SN-38-PLGA-MS group
(A), there were no significant differences between blank PLGA-MS group and the NS group. However, body weights of the mice in SN-38Sol group and SN-38-PLGA-MS group showed a stable increase, but the
Fig. 7. (A) The tumor volume versus time profiles; (B) Tumor volume of mice after 18 days of treatment in different preparation groups; (C) Tumor weight of mice after 18 days of treatment in different preparation groups. (D) Photograph of excised tumors, after treatment with NS, Blank MS, SN-38-Sol and SN-38-PLGA-MS. Data are expressed as mean ± SD, n = 8. Statistical significance was defined as **or ##p < 0.01 and ***p < 0.001. 7
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Fig. 8. (A) The body weight versus time profiles; Body weight of mice after 18 days of treatment NS, Blank MS, SN-38-Sol and SN-38-PLGA-MS. Data are expressed as mean ± SD, n = 8. Statistical significance was defined as **p < 0.01.
cannot inhibit tumor growth for a long time for its rapid release. However, in the SN-38-PLGA-MS group, most tumor cells were damaged with little ability of proliferation on the first day, but in the 6th day, severe necrosis was seen in central area of tumor cell the tumor cells with cell structure completely destroyed. And most tumor cells still on the 12th day were in necrosis as well as that of on the 6th day. But some of them began to proliferate on the 18th day. All the results obtained above indicated that the MS group has obvious sustained release effect, and can maintain the effective therapeutic concentration of the SN-38 at the tumor site for a long time comparing with the SN-38-Sol group. It can be concluded that SN-38-PLGA-MS had a stronger ability to suppress tumor growth than any other group. At the same time, the in vivo safety of the formulations administrated by intratumoral injection was evaluated by histopathological observation also. The H&E staining images of each tissue was shown in Fig. 10. There were no obvious differences in the cytoarchitecture of
compared with SN-38-Sol group (p < 0.01), which indicated SN-38PLGA-MS was a safe drug delivery system. 3.5.3. Results of histopathological analysis of tissues The light microscope images at 10X of tumor tissue after administration in different time of each group can be seen in Fig. 9. The dark purple area represents the proliferation of cells while the pink area means the cell has died or is dying. In the NS and the blank-PLGA-MS group, tumor cells were structurally intact and have strong proliferative capacity from the first day to the end, indicating that blank-PLGA-MS has no inhibitory effect on tumor growth. Whereas, in SN-38-Sol group, most of the tumor cells were damaged to broken flakes with lots of inflammatory cells infiltration on the first day. But on the 6th day, some tumors proliferated again. On the 18th day, the tumors proliferated faster, and it was no different from that without treatment, indicating that the SN-38-Sol
Fig. 9. H＆E stained images of tumors treated with NS, Blank PLGA MS, SN-38 Sol, SN-38-PLGA MS for tumor bearing mice at different times (10х). The scale bars were 200 μm. 8
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Fig. 10. H＆E stained images of main organs treated with NS, Blank PLGA MS, SN-38 Sol, SN-38-PLGA MS for tumor bearing mice at different times (10х). The scale bars were 200 μm.
microspheres should be carried out in a low temperature environment. In the part of pharmacokinetic studies, the normal KM mice were used as model animals. The concentrations determination of SN-38 in plasma and tumor samples was established by HPLC-UV method. After intratumoral injection, in the tumor, MRT and CL value for SN-38PLGA-MS group were 4.60 times and 0.22 times than the SN-38 solution group respectively, indicating that the retention time in tumor site of microspheres was extended significantly. While in the plasma, the Cmax in SN-38 solution group was 3.07 times than the SN-38-PLGA-MS group, which showed the higher leakage rate in the solution group. This is because molecules easily spread through the interstitial space after the intratumoral injection of the drug solution, so that the amount of the drug to maintain the local is small. Compared with the SN-38 solution, the release of SN-38 from microspheres is a slow process, which is helpful in increasing its tumor retention time. In vivo pharmacodynamic experiments were carried out using physiological saline as the negative control. The tumor volume of the NS group showed a rapid growth trend during the test period. 18-day test results showed that the mean tumor volume of MS group was the smallest. Subsequently, the tumor inhibition effect was quantitatively calculated. The results showed that the TGI, DT(d) and SGR (%/d) of SN-38-PLGA-MS were 1.42 times, 1.97 times and 0.51 times than those in SN-38-Sol, respectively, indicating that SN-38-PLGA-MS can better inhibit tumor growth compared with SN-38 solution. At the same time, compared with NS group, the mice body weight in SN-38-PLGA-MS
each tissue including heart, liver, spleen, lung, kidney and skin between the NS, blank-PLGA-MS, SN-38-Sol and the SN-38-PLGA-MS group. These results revealed that SN-38-PLGA-MS had good biocompatibility and low toxicity and was a promising candidate delivery carrier in our experimental conditions. 4. Discussion In this experiment, SN-38-PLGA-MS and free SN-38 were separated by ultra-low temperature centrifugation. Compared with the common methods such as dialysis, this method is simple to operate and has less drug leakage. Ultraviolet spectrophotometry was used to determine the drug content in SN-38-PLGA-MS. Because of the large amount of prescriptions, this method is simple, feasible and reliable. In this study, high-speed shearing under colostrum was carried out in the process of preparing SN-38-PLGA-MS, for the high-speed homogenizer could generate a large amount of heat at high speed, which had a certain influence on the stability of the system; If the temperature is much too high, the PLGA will become sticky and precipitate. In addition, low-temperature (4 °C) and high-speed centrifugation were also used in the centrifugal process of microspheres. It was found that if the SN-38-PLGA-MS suspension was centrifuged at the room temperature, the precipitated microspheres would turn into a pale yellow viscous rubbery aggregation and be difficult to be distracted in DMSO. In order to obtain SN-38-PLGA-MS, the steps of preparing and cleaning the 9
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group had increased, indicating that the SN-38-PLGA-MS prepared in this experiment had less toxic side effects. H&E slices of tumor tissue of mice showed that SN-38-PLGA-MS had a significant antitumor effect. H&E slices of the main organs showed that there were no significant differences between the SN-38-PLGA-MS group and the NS group, indicating that SN-38-PLGA-MS had no obvious side effects on the main organs of mice. All the results analyzed above showed that the SN-38-PLGA-MS prepared for introtumoral injection had certain clinical application potential.
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5. Conclusion In this research, SN-38-PLGA-MS was prepared successfully by emulsion solvent evaporation method and showed several favorable performances as a drug delivery system including spherical particles, narrow size distribution, high entrapment efficiency and drug loading. In addition, pharmacokinetic results showed that compared with SN38-Sol group, SN-38-PLGA-MS can obviously extend the retention time of SN-38 in the tumor site and decreased its leakage into plasma, which significantly improved the treatment effect on tumor and reduce the side effects of drugs. What's more, the pharmacodynamics and histopathological analysis of tumors in different times also showed that the SN-38-PLGA-MS exhibited a higher antitumor efficacy than any other groups. All of these results indicate that intratumoral injection of SN-38PLGA-MS is a promising and effective strategy for cancer therapeutic, which makes it possible for the effective treatment of the patients and provides strong research support for its extensive clinical applications. Conflicts of interest None. Declaration of interest The authors declares that there are no conflict of interests in this work. Acknowledgment The authors extend sincere gratitude to the Shandong University for the experiment platform needed to carry out this work. The authors also thank Guihua Huang for her mentoring advice and he selfless help of the lab team members. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jddst.2019.101178. References [1] M.D. Alvau, S. Tartaggia, A. Meneghello, B. Casetta, G. Calia, P.A. Serra, F. Polo, G. Toffoli, Enzyme-based electrochemical biosensor for therapeutic drug monitoring of anticancer drug irinotecan, Anal. Chem. 90 (10) (2018) 6012–6019 https://doi.org/10.1021/acs.analchem.7b04357. [2] G. Vassal, M.J. Terrier-Lacombe, M.C. Bissery, A.M. Venuat, F. Gyergyay, J. Benard, J. Morizet, I. Boland, P. Ardouin, B. Bressac-De-Paillerets, A. Gouyette, Therapeutic activity of CPT-11, a DNA-topoisomerase I inhibitor, against peripheral primitive neuroectodermal tumour and neuroblastoma xenografts, Br. J. Canc. 74 (4) (1996) 537–545 https://doi.org/10.1208/s12249-019-1327-x. [3] X. Yang, Y. Yang, Q. Jia, Y. Hao, J. Liu, G. Huang, Preparation and evaluation of irinotecan Poly(Lactic-co-Glycolic Acid) nanoparticles for enhanced anti-tumor therapy, AAPS. PharmSciTech 20 (3) (2019) 133–134 https://doi.org/10.1038/bjc. 1996.398. [4] A. Stein, W. Voigt, K. Jordan, Chemotherapy-induced diarrhea: pathophysiology, frequency and guideline-based management, Ther. Adv. Med. Oncol. 2 (1) (2010) 51–63 https://doi.org/10.1177/1758834009355164.
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