Drug Delivery
ISSN: 1071-7544 (Print) 1521-0464 (Online) Journal homepage: http://www.tandfonline.com/loi/idrd20
Ovarian cancer targeted hyaluronic acid-based nanoparticle system for paclitaxel delivery to overcome drug resistance Liping Wang & Erxia Jia To cite this article: Liping Wang & Erxia Jia (2015): Ovarian cancer targeted hyaluronic acidbased nanoparticle system for paclitaxel delivery to overcome drug resistance, Drug Delivery, DOI: 10.3109/10717544.2015.1101792 To link to this article: http://dx.doi.org/10.3109/10717544.2015.1101792
Published online: 04 Nov 2015.
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Date: 27 November 2015, At: 03:57
http://informahealthcare.com/drd ISSN: 1071-7544 (print), 1521-0464 (electronic) Drug Deliv, Early Online: 1–8 ! 2015 Taylor & Francis. DOI: 10.3109/10717544.2015.1101792
Ovarian cancer targeted hyaluronic acid-based nanoparticle system for paclitaxel delivery to overcome drug resistance Liping Wang and Erxia Jia
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Department of Gynecology and Obstetrics, The Fourth People’s Hospital of Ji’nan, Ji’nan, Shandong, People’s Republic of China
Abstract
Keywords
Objective: Most primary human ovarian tumors and peritoneal implants, as well as tumor vascular endothelial cells, express the CD44 family of cell surface proteoglycans, the natural ligand for which is hyaluronic acid (HA). Paclitaxel (PTX) is an effective chemotherapeutic agent that is widely used for the treatment of several cancers, including ovarian cancer. This study aimed to develop a HA-based PTX-loaded nanoparticle system to improve the ovarian cancer therapeutic effects. Methods: PTX-loaded cationic nanostructured lipid nanoparticles (PTX-NLCs) were prepared. HA-PE was then coated onto the PTX-NLCs by electrostatic adsorption to form HA-PTX-NLCs. In vitro tumor cell inhibition efficiency was analyzed on SKOV3 human ovarian cancer cells (SKOV3 cells) and PTX-resistant SKOV3 cells (SKOV3/PTX cells). In vivo anticancer ability was evaluated with mice bearing SKOV3 ovarian cancer cells xenografts. Results: HA-PTX-NLCs had an average diameter of 163 nm, and PTX was incorporated with an efficiency of over 80%. The in vitro viability of SKOV3 cells and SKOV3/PTX cells was obviously inhibited by HA-PTX-NLCs. In the ovarian cancer cells model, significant reduction in tumor growth was observed, whereas the conventional PTX injection group did not achieve significance. Conclusion: This study demonstrated that significantly improved results were obtained by the newly constructed HA-PTX-NLCs, in terms of in vitro and in vivo therapeutic efficacy. These findings strongly support the superiority of HA based nano-system for the PTX delivery, thus enhance the efficacy of ovarian cancer chemotherapy.
Drug resistance, hyaluronic acid, lipid nanoparticles, ovarian cancer, paclitaxel delivery
Introduction Ovarian cancer is the leading cause of death from gynecologic cancer in the US and it is the country’s fifth most common cause of cancer mortality in women. In 2014, it is estimated that 21 980 new diagnoses and 14 270 deaths from this neoplasm will occur in the US (Siegel et al., 2014). Primary treatment for presumed ovarian cancer consists of appropriate surgical staging and cytoreduction, followed in most patients by systemic chemotherapy. Currently, the firstline therapy accepted by a consensus of the NCCN panel includes carboplatin, paclitaxel (PTX) or carboplatin and paclitaxel. Although the response rate to PTX and platinum is up to 80%, more than 85% eventually relapse because of the emergence of multidrug resistance (MDR) (Markman, 2008; Zahedi et al., 2012; Coleman et al., 2013; Lheureux et al., 2015). MDR remains one of the most significant challenges to cure ovarian cancer (Lopez et al., 2013; Yang et al., 2015). Paclitaxel (PTX), a microtubule-stabilizing agent, is indicated for different types of cancers including ovarian Address for correspondence: Erxia Jia, Department of Gynecology and Obstetrics, The Fourth People’s Hospital of Ji’nan, 50 Shifan Road, Tianqiao District, Ji’nan, 250012, Shandong Province, People’s Republic of China. Email: [email protected]
History Received 24 August 2015 Revised 24 September 2015 Accepted 26 September 2015
cancer, breast cancer, non-small cell lung cancer, etc. In order to improve the outcome of PTX for ovarian cancer, strategies to circumvent MDR have been studied extensively in recent years (Sarisozen et al., 2012; Lopez et al., 2013). With the development of nanotechnology, drug delivery nanosystems, especially adding a targeting moiety to the surface of nanocarriers, have shown great promise to overcome MDR and improve the chemotherapeutical efficacy (Liu et al., 2014; Liang et al., 2015). Various targeting molecules have been applied to target PTX to tumor tissues, such as folic acid, biotin, hyaluronic acid (HA) (Yang et al., 2013; Zhang et al., 2014; Yang et al., 2015). HA is an anionic, non-sulfated glycosaminoglycan. HA has an important biological role in the cell adhesion, migration, invasion, proliferation, differentiation and angiogenesis by binding to cell specific receptors like glycoprotein CD44 and receptor for HA-mediated motility (RHAMM) (Ghosh et al., 2012; Saadat et al., 2014). The presence of high expression levels of CD44 was considered to be associated with drug resistance and is an unfavorable prognosis or survival marker in cancers including ovarian cancer, lung cancer, etc (Yim & Na, 2010; Cho et al., 2011; Shi et al., 2013; Liu et al., 2014; Liang et al., 2015). HA based nanoparticle systems can be obtained by covalent bond
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modification or electrostatic attraction. In this study, hyaluronic acid (HA)-1,2-distearoyl phosphatidylethanolamine (DSPE) was synthesized, and HA-DSPE coated PTX-loaded cationic lipid nanoparticles were engineered. Nano-sized drug delivery systems (NDDS) are ideal for i.v. administration as they can take advantage of the enhanced permeation and retention (EPR) effect, and have been shown to overcome MDR mechanisms by directing endocytosismediated cellular internalization of drug and/or interacting directly with efflux pumps (Zahedi et al., 2012). Lipid nanoparticles are core-shell nanoparticle comprising polymer cores and lipid/lipid-PEG shells. The advantages of lipid nanoparticles are the followings: biocompatibility and biodegradability; controlled drug release; higher physical stability in vitro and in vivo; passive and active targeting; and capable of large-scale production (Hadinoto et al., 2013). In our previous study, folate-modified, cisplatin-loaded lipid carriers were constructed for cervical cancer chemotherapy (Zhang et al., 2015a,b). Furthermore, the lipid shell of lipid nanoparticles made them easier to be modified by HA-DSPE. Therefore, lipid nanoparticles may be the ideal nanocarrier as the delivery system for PTX (the hydrophobic anticancer drug). In this study, PTX-loaded lipid nanoparticles (PTX-NLCs) were prepared. HA-DSPE was synthesized and then PTXNLCs were modified to form HA-PTX-NLCs. Physical parameters such as mean diameters, drug loading efficiency, etc. In vitro tumor cell inhibition efficiency was analyzed on SKOV3 human ovarian cancer cells (SKOV3 cells) and PTXresistant SKOV3 cells (SKOV3/PTX cells). In vivo anticancer ability was evaluated with mice bearing SKOV3 ovarian cancer cells xenografts. HA-PTX-NLCs were anticipated to be the ideal platform to enhance therapeutic efficacy and overcome drug resistance.
Materials and methods Materials, cells and animal Paclitaxel (PTX) was obtained from Hisun Pharmaceutical Co Ltd. (Taizhou, China). Hyaluronic acid (HA, MW 300 kDa) was provided by Shandong Freda Biochem Co., Ltd. (China). Soya lecithin was obtained from Shanghai Advanced Vehicle Technology Co., Ltd. (Shanghai, China). Soybean oil was purchased from Guangzhou Hanfang Pharmaceutical Co., Ltd. (Guangzhou, China). 1,2-Distearoyl phosphatidylethanolamine (DSPE, C41H82NO8P) were purchased from Lipoid GmbH (Ludwigshafen, Germany). Tween-80, and stearic acid, N-Hydroxysuccinimide (NHS) and 1-ethyl-3-(3dimethylamino-propyl) carbodiimide (EDC) and pyrene
Figure 1. Scheme diagram to illustrate the preparation of HA-PTX-NLCs.
Drug Deliv, Early Online: 1–8
were purchased from Sigma Aldrich (St. Louis, MO). All other chemicals and reagents used were of cell culture or reagent grade. SKOV3 cells were obtained from the American type culture collection (Manassas, VA). BALB/c nude mice (6–8 weeks old, 20–25 g weight) were purchased from Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China). Synthesis of HA-DSPE Hyaluronic acid-1,2-distearoyl phosphatidylethanolamine (HA-DSPE) was synthesized using the similar method described by our group previously, with modification (Zhang et al., 2015a). Briefly, 100 mg of HA was dissolved in deionized water, then two equivalence of EDC and NHS were added into the HA solution. Five equivalence of DSPE was dissolved in dichloromethane (DCM) and triethylamine and added to HA solution. The mixture was stirred for 24 h under nitrogen. Finally, HA-DSPE was obtained and purified by dialyzing for 48 h. Structure confirmation: IR /cm1: 3311 (–NH–); 2972(–CH2–); 1741(–CO–); 1603(–NH–CO–); 1321 (–NH–CH2–); 768(–NH–). 1H NMR (DMSO-d6, 300 MHz), d 9.82 (s, –NH–CO–); 8.11 (d, –CH–); 6.53 (t, –CH–); 4.28 (m, –CH2–); 2.41 (t, –CO–CH2–); 0.93 (t, –CH3). The production rate of the HA-DSPE was 75.83%. Preparation of PTX-NLCs Paclitaxel (PTX)-NLCs were prepared by emulsification method (Figure 1) (Andey et al., 2015). Oil phase was composed of stearic acid, soya lecithin and soybean oil. Aqueous phase was formed by Tween-80 (1%) and deionized water. The PTX was dissolved in acetone, added into oil phase and warmed to 70 C. The aqueous phase warmed to 70 C, and then added to the oil phase under high speed homogenizing (30 000 rpm; 15 min). The resulting PTXNLCs were purified by dialysis against pH 7.4 phosphate buffered saline (PBS) for 4 h, centrifuged for 15 min at 5000 rpm, lyophilized and kept for further analysis. Preparation of HA-PTX-NLCs Hyaluronic acid (HA)-PTX-NLCs were prepared using the same method as the preparation of PTX-NLCs, except for the HA-DSPE was dissolved in aqueous phase warmed to 65 C (Figure 1). To select the appropriate HA-DSPE amount for the formulation, different ratio of HA-DSPE to PTX-NLCs (1:3, 1:2, 1:1, 2:1, 3:1, w/w) were used, and the property of the resulting HA-PTX-NLCs were analyzed and the best ratio was determined.
DOI: 10.3109/10717544.2015.1101792
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Characterization of drug loaded NLCs
Cell viability assays in vitro
Particle size and zeta potential
SKOV3 cells or SKOV3/PTX cells were plated at a density of 1 104 cells/well in 96-well plates. Cells were cultured in DMEM medium (supplemented with 10% fetal bovine serum, 100 U/mL of penicillin and 100 mg/mL streptomycin) and maintained at 37 C in a 5% CO2 atmosphere. After the overnight incubation, the medium was replaced with medium containing HA-PTX-NLCs, PTX-NLCs, PTX solution, blank NLCs. Then the cells were incubated at 37 C, 5% CO2 for additional 72 h. The cell viability was measured using the 3 -(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) method according to manufacturer’s instructions.
The measures of mean particle size and polydispersity index (PDI) of different NLCs formulations were performed by dynamic light scattering technique using a Zetasizer (NanoZS90, Malvern Instruments, UK). The zeta potential was measured by the nanoparticles electrophoresis mobility using a U-type tube at 20 C. Drug entrapment efficiency and loading content
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Hyaluronic acid based nanoparticle system
Entrapment efficiency (EE) and loading content (LC) of HAPTX-NLCs and PTX-NLCs were determined were determined using a subtraction method (Zhang et al., 2015b). Briefly, drug-loaded NLCs were dispersed in phosphate buffer solution (2% Tween 80 in PBS, pH 7.4) and vortexed for 3 min to dissolve the free PTX. Then, the dispersion was centrifuged for 0.5 h at 10 000 rpm to separate the free drug from NLCs. After the centrifugation, PTX content in the supernatant was measured by HPLC (Waters2695, Milford, MA). A 20 mL of the solution was injected into an InertsilÕ ODS-3 V analytical column. Mobile phase was consisted of a mixture of acetonitrile and water (50:50, v/v). Flow rate was kept at 1.0 mL/min and system was maintained at 35 C, the detection was carried out at ¼ 227 nm. ðTotal weight of PTX added Weight of free PTXÞ EEð%Þ ¼ Total weight of PTX added 100 ðTotal weight of PTX added Weight of free PTXÞ Total weight of PTX and NLCs 100
LCð%Þ ¼
Drug release study in vitro Paclitaxel (PTX) release from HA-PTX-NLCs, PTX-NLCs and PTX solution was assessed by the dialysis method in PBS 5.5 and 7.4, respectively. Briefly, the NLCs were suspended in 10 mL of the PBS release medium in dialysis membrane (molecular weight cutoff: 10–14 kDa) and transferred to a glass beaker containing 90 ml of PBS (pH 7.4). The samples were incubated at 37 ± 0.5 C with constant agitation of 200 rpm for the release studies. One milliliter of buffer was periodically withdrawn and replaced with equal volume of fresh PBS. The amount of PTX released was determined by HPLC as describe in the above ‘‘EE and LC’’ part. In vitro Stability of HA-PTX-NLCs The stability of HA-PTX-NLCs in 50% fetal bovine serum (FBS) was evaluated by measuring their particle size, PDI and zeta potential after incubation at 37 C for 24 h. At scheduled times (24 h), 1 mL of each sample was diluted with 2 mL THF and the mixture was bath sonicated for 5 min, followed by centrifugation at 10 000r/min for 5 min. PTX content in the supernatant was measured by HPLC method describe in the above ‘‘EE and LC’’ section.
Antitumor efficiency studies in vivo BALB/c mice were subcutaneously inoculating with SKOV3 cells suspended in PBS. When tumor volume reached about 50 mm3, transplanted mice were randomly divided into five groups containing six animals each. HA-PTX-NLCs, PTXNLCs, PTX solution, blank NLCs and normal saline (NS) were injected every six days, respectively. Tumor length and width were measured every 3 days by digital vernier caliper to depict the late stage of the tumor progression. The tumor volume (TV) was calculated by the following equation: TV ¼ Length Width2 =2 The antitumor efficacy of each formulation was evaluated by tumor inhibition rate (TIR), which was calculated using the following equation: Tumor weight of sample group Tumor weight of control group TIRð%Þ ¼ Tumor weight of control group 100 The body weight changes of different groups were also recorded. Biodistribution investigation BALB/c mice were treated the same way mentioned in the ‘‘Antitumor efficiency studies in vivo’’ section. At predetermined time intervals, mice were sacrificed and the tumor, heart, liver, spleen, lung and kidney of mice were collected and stored. To determine the amount of PTX in each tissue, tissues were first weighed and homogenized with physiological saline. Then, PTX was extracted with acetonitrile and methanol (1:1, v/v) and centrifuged (15 000 rpm, 10 min). The supernatant was collected, and the amount of PTX was determined by HPLC as describe in the above ‘‘EE and LC’’ section. Statistical analysis All studies were repeated three times and all measurements were carried out in triplicate. Results were reported as means ± SD (SD ¼ standard deviation). Statistical significance was preformed by two-tailed Student’s t-test at p50.05.
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Results and discussion
Table 1. Characterization of different vectors.
Characterization of drug loaded NLCs
Samples
Blank NLCs
PTX-NLCs
HA-PTX-NLCs
Lipid nanoparticles can take advantage of the enhanced permeation and retention (EPR) effect, and have been shown to overcome MDR mechanisms by directing endocytosis-mediated cellular internalization of drug and/or interacting directly with efflux pumps (Hadinoto et al., 2013; Nandini et al., 2015). Size and distribution are important factors for the nanocarriers, which can influence the efficiency of the drug loaded nanoparticles (Jin et al., 2007; Nandini et al., 2015). Particle size around 100 nm could decreases uptake by the liver, prolongs circulation time in the blood and improves bioavailability. The mean particle size of HA-PTX-NLCs, PTX-NLCs, blank NLCs was 114.3, 96.1 and 93.6 nm, respectively (Table 1). No significant change in diameter was found from blank NLC and PTX-NLCs; however, the size of HA-PTX-NLCs was larger due to the coating of the HA ligands. The zeta potential is a key factor for the stability of nanosystems (Xu et al., 2009). The positive charge of the NLCs and also facilitate the delivery of drug to the negatively charged cancer cells. The zeta potential of HA-PTX-NLCs, PTXNLCs and blank NLCs was +11.5, +29.6 and +17.9 mV, respectively. Higher zeta potential of PTX-NLCs than blank NLCs may contribute to the positively charged PTX. The zeta potential of HA-PTX-NLCs were the lowest, due to the negatively charged HA neutralized the charge of the cationic NLCs. The EE of HA-PTX-NLCs, PTX-NLCs was over 80%, and the LC was 5.6% for PTX-NLCs and 3.2% for HA-PTX-NLCs.
Particle size (nm) Size distribution Zeta potential (mV) EE (%) LC (%)
93.6 ± 3.1 0.10 ± 0.02 +17.9 ± 2.3 N/A N/A
96.1 ± 3.4 0.13 ± 0.02 +29.6 ± 3.4 83.1 ± 3.2 5.6 ± 0.6
114.3 ± 4.6 0.16 ± 0.03 +11.5 ± 1.7 81.9 ± 2.8 3.2 ± 0.5
Table 2. Optimization of HA-PTX-NLCs. HA-DSPE to PTX-NLCs ratio (w/w) 1:3 1:2 1:1 2:1 3:1
Zeta potential (mV)
Particle size (nm)
EE (%)
+24.3 ± 4.1 +18.6 ± 2.8 +11.7 ± 1.9 +10.1 ± 2.6 +9.9 ± 2.2
99.7 ± 3.4 105.3 ± 3.7 113.4 ± 4.6 237.6 ± 45.2 339.9 ± 64.7
82.3 ± 2.6 81.7 ± 2.7 82.4 ± 2.4 61.2 ± 6.4 38.3 ± 8.2
Optimization of HA-PTX-NLCs Hyaluronic acid (HA) is an anionic glycosaminoglycan. HA has an important biological role in the cell adhesion, migration, invasion, proliferation, differentiation and angiogenesis by binding to cell specific receptors like glycoprotein CD44, who was considered to be associated with drug resistance and is an unfavorable prognosis or survival marker in cancers including ovarian cancer (Ghosh et al., 2012; Saadat et al., 2014). To optimize the best HA-PTXNLCs formulation, different ratio of HA-DSPE to PTXNLCs (1:3, 1:2, 1:1, 2:1, 3:1, w/w) were used, and the property of the resulting HA-PTX-NLCs were analyzed (Table 2). The property of the HA-PTX-NLCs would be changed as follows due to the adding of HA ligands: (1) the presence of anionic HA could neutralize the positive potential of the cationic NLCs, cause the change of potential. (2) The HA-DSPE could be the shell of the HAPTX-NLCs, thus increased the particle size. It is worth noting that the exclusive using of HA ligands may cause the leap of the size and the wide size distribution. (3) The EE should be maintained at a high level; significantly decrease of EE may be the evidence of the instable of the NLCs system. We can tell from Table 2 that the ratio at 1:1 of HA-DSPE to PTX-NLCs was best for all the parameters of HA-PTXNLCs, including stable zeta potential and EE, suitable particle size and narrow size distribution.
Figure 2. PTX release from HA-PTX-NLCs, PTX-NLCs, and PTX solution in PBS 7.4.
Drug release study in vitro Paclitaxel (PTX) release from HA-PTX-NLCs and PTXNLCs was assessed in PBS 7.4 (Figure 2) and 5.5 (Figure 3), respectively. Figures 2 and 3 shown that: (1) PTX release from both HA-PTX-NLCs and PTX-NLCs were faster in the acidic (pH 5.5) than neutral environment (pH 7.4), this could be the evidence that the drug would release more easily in acidic tumor tissue; (2) PTX release from HA-PTX-NLCs were slower than PTX-NLCs, this may due the HA appearing on the surface that delayed the release of drugs. The release profile shows that HA-PTX-NLCs formulation has the capacity to release PTX at a sustained rate, let the anticancer drug to maintain continuous efficiency. In vitro Stability of HA-PTX-NLCs Considering the intravenous administration usage of the drug loaded NLCs, the stability of HA-PTX-NLCs and PTX-NLCs
Hyaluronic acid based nanoparticle system
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Figure 5. Cell viability of HA-PTX-NLCs, PTX-NLCs and PTX solution in SKOV3/PTX cells. Figure 3. PTX release from HA-PTX-NLCs, PTX-NLCs, and PTX solution in PBS 5.0.
Table 3. Stability of HA-PTX-NLCs in 50% FBS.
Samples HA-PTX-NLCs PTX-NLCs
Table 4. IC50 of HA-PTX-NLCs, PTX-NLCs, PTX solution evaluated in SKOV3 cells and SKOV3/PTX cells. Samples
Particle size (nm)
Size distribution
Zeta potential (mV)
118.7 ± 5.9 101.4 ± 4.2
0.19 ± 0.05 0.15 ± 0.03
17.6 ± 2.4 8.6 ± 0.9
Figure 4. Cell viability of HA-PTX-NLCs, PTX-NLCs, and PTX solution in SKOV3 cells.
in serum was investigated to check for aggregation in the presence of proteins. The particle size, size distribution, and zeta potential are described in Table 3. The size and distribution of HA-PTX-NLCs and PTX-NLCs were slightly increased after incubation with serum, but data shown no significant changes (p40.05). The zeta potential of the NLCs in the presence of serum was inversed from positive
SKOV3 cells (mM) SKOV3/PTX cells (mM)
HA-PTX-NLCs
PTX-NLCs
Free PTX
0.011 ± 0.003 0.019 ± 0.004
0.024 ± 0.006 0.045 ± 0.008
0.216 ± 0.031 23.156 ± 2.351
Figure 6. The tumor growth inhibition effect of different groups.
Table 5. Tumor inhibition rates of samples in ovarian cancer bearing mice. Samples
HA-PTX-NLCs
PTX-NLCs
Free PTX
TIR (%)
85.0 ± 4.1
72.6 ± 3.3
25.1 ± 1.9
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to negative. This phenomenon may due to the negatively charged serum that changed the surface charge of the positive NLCs (Le Broc-Ryckewaert et al., 2013). The relatively stable characteristic illustrated that HA-PTX-NLCs and PTX-NLCs were stable in serum and would not aggregate after intravenous administration.
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Cell viability assays in vitro
Figure 7. Body weight changes of different groups.
Figure 8. Mean tissue concentrations of PTX after injection of PTX solution.
Figure 9. Mean tissue concentrations of PTX after injection of HA-PTX-NLCs.
In vitro SKOV3 and SKOV3/PTX cells cytotoxicity was evaluated by MTT assay to evaluate the activity of drug loaded NLCs against ovarian cancer cells. Figures 4 and 5 showed the HA-PTX-NLCs, PTX-NLCs and PTX solution reduced cell viability in a concentration-dependent manner. At all the studied PTX concentrations, the cytotoxicity of HAPTX-NLCs was higher than PTX-NLCs (p50.05), tumor cell inhibition effect of drug loaded NLCs was better than free PTX solution (p50.05). The IC50 values of HA-PTXNLCs, PTX-NLCs and PTX solution were presented in Table 4. PTX-NLCs significantly decreased the IC50 value 10 times compared to PTX solution (p50.05), implying that NLCs formulations show higher cytotoxicity against ovarian cancer cells. HA-PTX-NLCs decreased the IC50 value two times dose advantage on PTX-NLCs (p50.05). The enhanced efficacy of PTX loaded NLCs against ovarian tumor cells may be explained mechanism that the enhanced intracellular drug accumulation by ligand-receptor recognition, and by nanosized carrier uptake (Liu et al., 2011).
Hyaluronic acid based nanoparticle system
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DOI: 10.3109/10717544.2015.1101792
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Antitumor efficiency studies in vivo
References
In vivo antitumor efficiency of NLCs was evaluated in mice bearing ovarian cancer xenograft. The tumor growth inhibition effect was shown in Figure 6 in terms of the tumor volume changes. The TIR of different groups were illustrated in Table 5. HA-PTX-NLCs group exhibited the highest TIR (85%), followed by PTX-NLCs (73%) and PTX solution (25%). The curves of tumor volume and TIR showed that: (1) obvious tumor regression was observed in mice treated with HA-PTX-NLCs, PTX-NLCs, PTX solution, while no antitumor effect was observed in the blank NLCs and NS groups; (2) tumor growth was more significantly inhibited by drug loaded NLCs groups (HAPTX-NLCs and PTX-NLCs) than the PTX solution (p50.05); (3) HA-PTX-NLCs showed better anti-tumor efficacy than PTX-NLCs (p50.05), the most obviously tumor regressions were observed in the HA-PTX-NLCs group, the tumor growth was prominently inhibited than any other group (p50.05). Body weight changes were illustrated in Figure 7. Obviously decrease in mean body weights were found in blank NLCs and NS group, with the reduced foods intake, moving inactively. However, slightly increase of body weights were found in HA-PTX-NLCs and PTX-NLCs, the foods intake and movement is normal. These results may suggest the less systemic toxic side effect of the NLCs formulations for the in vivo cancer treatment.
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Biodistribution investigation Figure 8 showed that the concentration of PTX was higher in the heart and kidney for the PTX solution group, which may explain the adverse side effects. However, the concentrations of PTX in the heart and kidney were lower for the HA-PTX-NLCs group (Figure 9), which was expected to reduce the side effects. The concentration of PTX for the HA-PTX-NLCs group was higher in the tumor and liver compared with the drugs solution group, which may be due to the size of NLCs and the EPR effect of nanocarriers in the tumor. Higher concentrations of HAPTX-NLCs were found in tumor tissue during 12–48 h after intravenous injection, shown the sustained release behavior of the HA-PTX-NLCs.
Conclusions For ovarian cancer chemotherapy, SKOV3 cells and mice bearing ovarian cancer model were used to evaluate the efficiency of HA based, PTX loaded NLCs system. The results suggest that this drug delivery system could be stable in serum, achieve targeted delivery of drug, reduce the systemic toxicity and reach the outstanding antitumor efficiency. These findings strongly support the superiority of HA based nano-system for the delivery of drugs and for the targeted treatment of ovarian cancer.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
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Zhang G, Liu F, Jia E, et al. (2015a). Folatemodified, cisplatin-loaded lipid carriers for cervical cancer chemotherapy. Drug Deliv Jul 13:1–5. [Epub ahead of print]. DOI: 10.3109/10717544.2015.1054052. Zhang W, Li C, Shen C, et al. (2015b). Prodrug-based nano-drug delivery system for co-encapsulate paclitaxel and carboplatin for lung
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ISSN: 1071-7544 (Print) 1521-0464 (Online) Journal homepage: http://www.tandfonline.com/loi/idrd20
Ovarian cancer targeted hyaluronic acid-based nanoparticle system for paclitaxel delivery to overcome drug resistance Liping Wang & Erxia Jia To cite this article: Liping Wang & Erxia Jia (2015): Ovarian cancer targeted hyaluronic acidbased nanoparticle system for paclitaxel delivery to overcome drug resistance, Drug Delivery, DOI: 10.3109/10717544.2015.1101792 To link to this article: http://dx.doi.org/10.3109/10717544.2015.1101792
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http://informahealthcare.com/drd ISSN: 1071-7544 (print), 1521-0464 (electronic) Drug Deliv, Early Online: 1–8 ! 2015 Taylor & Francis. DOI: 10.3109/10717544.2015.1101792
Ovarian cancer targeted hyaluronic acid-based nanoparticle system for paclitaxel delivery to overcome drug resistance Liping Wang and Erxia Jia
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Department of Gynecology and Obstetrics, The Fourth People’s Hospital of Ji’nan, Ji’nan, Shandong, People’s Republic of China
Abstract
Keywords
Objective: Most primary human ovarian tumors and peritoneal implants, as well as tumor vascular endothelial cells, express the CD44 family of cell surface proteoglycans, the natural ligand for which is hyaluronic acid (HA). Paclitaxel (PTX) is an effective chemotherapeutic agent that is widely used for the treatment of several cancers, including ovarian cancer. This study aimed to develop a HA-based PTX-loaded nanoparticle system to improve the ovarian cancer therapeutic effects. Methods: PTX-loaded cationic nanostructured lipid nanoparticles (PTX-NLCs) were prepared. HA-PE was then coated onto the PTX-NLCs by electrostatic adsorption to form HA-PTX-NLCs. In vitro tumor cell inhibition efficiency was analyzed on SKOV3 human ovarian cancer cells (SKOV3 cells) and PTX-resistant SKOV3 cells (SKOV3/PTX cells). In vivo anticancer ability was evaluated with mice bearing SKOV3 ovarian cancer cells xenografts. Results: HA-PTX-NLCs had an average diameter of 163 nm, and PTX was incorporated with an efficiency of over 80%. The in vitro viability of SKOV3 cells and SKOV3/PTX cells was obviously inhibited by HA-PTX-NLCs. In the ovarian cancer cells model, significant reduction in tumor growth was observed, whereas the conventional PTX injection group did not achieve significance. Conclusion: This study demonstrated that significantly improved results were obtained by the newly constructed HA-PTX-NLCs, in terms of in vitro and in vivo therapeutic efficacy. These findings strongly support the superiority of HA based nano-system for the PTX delivery, thus enhance the efficacy of ovarian cancer chemotherapy.
Drug resistance, hyaluronic acid, lipid nanoparticles, ovarian cancer, paclitaxel delivery
Introduction Ovarian cancer is the leading cause of death from gynecologic cancer in the US and it is the country’s fifth most common cause of cancer mortality in women. In 2014, it is estimated that 21 980 new diagnoses and 14 270 deaths from this neoplasm will occur in the US (Siegel et al., 2014). Primary treatment for presumed ovarian cancer consists of appropriate surgical staging and cytoreduction, followed in most patients by systemic chemotherapy. Currently, the firstline therapy accepted by a consensus of the NCCN panel includes carboplatin, paclitaxel (PTX) or carboplatin and paclitaxel. Although the response rate to PTX and platinum is up to 80%, more than 85% eventually relapse because of the emergence of multidrug resistance (MDR) (Markman, 2008; Zahedi et al., 2012; Coleman et al., 2013; Lheureux et al., 2015). MDR remains one of the most significant challenges to cure ovarian cancer (Lopez et al., 2013; Yang et al., 2015). Paclitaxel (PTX), a microtubule-stabilizing agent, is indicated for different types of cancers including ovarian Address for correspondence: Erxia Jia, Department of Gynecology and Obstetrics, The Fourth People’s Hospital of Ji’nan, 50 Shifan Road, Tianqiao District, Ji’nan, 250012, Shandong Province, People’s Republic of China. Email: [email protected]
History Received 24 August 2015 Revised 24 September 2015 Accepted 26 September 2015
cancer, breast cancer, non-small cell lung cancer, etc. In order to improve the outcome of PTX for ovarian cancer, strategies to circumvent MDR have been studied extensively in recent years (Sarisozen et al., 2012; Lopez et al., 2013). With the development of nanotechnology, drug delivery nanosystems, especially adding a targeting moiety to the surface of nanocarriers, have shown great promise to overcome MDR and improve the chemotherapeutical efficacy (Liu et al., 2014; Liang et al., 2015). Various targeting molecules have been applied to target PTX to tumor tissues, such as folic acid, biotin, hyaluronic acid (HA) (Yang et al., 2013; Zhang et al., 2014; Yang et al., 2015). HA is an anionic, non-sulfated glycosaminoglycan. HA has an important biological role in the cell adhesion, migration, invasion, proliferation, differentiation and angiogenesis by binding to cell specific receptors like glycoprotein CD44 and receptor for HA-mediated motility (RHAMM) (Ghosh et al., 2012; Saadat et al., 2014). The presence of high expression levels of CD44 was considered to be associated with drug resistance and is an unfavorable prognosis or survival marker in cancers including ovarian cancer, lung cancer, etc (Yim & Na, 2010; Cho et al., 2011; Shi et al., 2013; Liu et al., 2014; Liang et al., 2015). HA based nanoparticle systems can be obtained by covalent bond
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modification or electrostatic attraction. In this study, hyaluronic acid (HA)-1,2-distearoyl phosphatidylethanolamine (DSPE) was synthesized, and HA-DSPE coated PTX-loaded cationic lipid nanoparticles were engineered. Nano-sized drug delivery systems (NDDS) are ideal for i.v. administration as they can take advantage of the enhanced permeation and retention (EPR) effect, and have been shown to overcome MDR mechanisms by directing endocytosismediated cellular internalization of drug and/or interacting directly with efflux pumps (Zahedi et al., 2012). Lipid nanoparticles are core-shell nanoparticle comprising polymer cores and lipid/lipid-PEG shells. The advantages of lipid nanoparticles are the followings: biocompatibility and biodegradability; controlled drug release; higher physical stability in vitro and in vivo; passive and active targeting; and capable of large-scale production (Hadinoto et al., 2013). In our previous study, folate-modified, cisplatin-loaded lipid carriers were constructed for cervical cancer chemotherapy (Zhang et al., 2015a,b). Furthermore, the lipid shell of lipid nanoparticles made them easier to be modified by HA-DSPE. Therefore, lipid nanoparticles may be the ideal nanocarrier as the delivery system for PTX (the hydrophobic anticancer drug). In this study, PTX-loaded lipid nanoparticles (PTX-NLCs) were prepared. HA-DSPE was synthesized and then PTXNLCs were modified to form HA-PTX-NLCs. Physical parameters such as mean diameters, drug loading efficiency, etc. In vitro tumor cell inhibition efficiency was analyzed on SKOV3 human ovarian cancer cells (SKOV3 cells) and PTXresistant SKOV3 cells (SKOV3/PTX cells). In vivo anticancer ability was evaluated with mice bearing SKOV3 ovarian cancer cells xenografts. HA-PTX-NLCs were anticipated to be the ideal platform to enhance therapeutic efficacy and overcome drug resistance.
Materials and methods Materials, cells and animal Paclitaxel (PTX) was obtained from Hisun Pharmaceutical Co Ltd. (Taizhou, China). Hyaluronic acid (HA, MW 300 kDa) was provided by Shandong Freda Biochem Co., Ltd. (China). Soya lecithin was obtained from Shanghai Advanced Vehicle Technology Co., Ltd. (Shanghai, China). Soybean oil was purchased from Guangzhou Hanfang Pharmaceutical Co., Ltd. (Guangzhou, China). 1,2-Distearoyl phosphatidylethanolamine (DSPE, C41H82NO8P) were purchased from Lipoid GmbH (Ludwigshafen, Germany). Tween-80, and stearic acid, N-Hydroxysuccinimide (NHS) and 1-ethyl-3-(3dimethylamino-propyl) carbodiimide (EDC) and pyrene
Figure 1. Scheme diagram to illustrate the preparation of HA-PTX-NLCs.
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were purchased from Sigma Aldrich (St. Louis, MO). All other chemicals and reagents used were of cell culture or reagent grade. SKOV3 cells were obtained from the American type culture collection (Manassas, VA). BALB/c nude mice (6–8 weeks old, 20–25 g weight) were purchased from Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China). Synthesis of HA-DSPE Hyaluronic acid-1,2-distearoyl phosphatidylethanolamine (HA-DSPE) was synthesized using the similar method described by our group previously, with modification (Zhang et al., 2015a). Briefly, 100 mg of HA was dissolved in deionized water, then two equivalence of EDC and NHS were added into the HA solution. Five equivalence of DSPE was dissolved in dichloromethane (DCM) and triethylamine and added to HA solution. The mixture was stirred for 24 h under nitrogen. Finally, HA-DSPE was obtained and purified by dialyzing for 48 h. Structure confirmation: IR /cm1: 3311 (–NH–); 2972(–CH2–); 1741(–CO–); 1603(–NH–CO–); 1321 (–NH–CH2–); 768(–NH–). 1H NMR (DMSO-d6, 300 MHz), d 9.82 (s, –NH–CO–); 8.11 (d, –CH–); 6.53 (t, –CH–); 4.28 (m, –CH2–); 2.41 (t, –CO–CH2–); 0.93 (t, –CH3). The production rate of the HA-DSPE was 75.83%. Preparation of PTX-NLCs Paclitaxel (PTX)-NLCs were prepared by emulsification method (Figure 1) (Andey et al., 2015). Oil phase was composed of stearic acid, soya lecithin and soybean oil. Aqueous phase was formed by Tween-80 (1%) and deionized water. The PTX was dissolved in acetone, added into oil phase and warmed to 70 C. The aqueous phase warmed to 70 C, and then added to the oil phase under high speed homogenizing (30 000 rpm; 15 min). The resulting PTXNLCs were purified by dialysis against pH 7.4 phosphate buffered saline (PBS) for 4 h, centrifuged for 15 min at 5000 rpm, lyophilized and kept for further analysis. Preparation of HA-PTX-NLCs Hyaluronic acid (HA)-PTX-NLCs were prepared using the same method as the preparation of PTX-NLCs, except for the HA-DSPE was dissolved in aqueous phase warmed to 65 C (Figure 1). To select the appropriate HA-DSPE amount for the formulation, different ratio of HA-DSPE to PTX-NLCs (1:3, 1:2, 1:1, 2:1, 3:1, w/w) were used, and the property of the resulting HA-PTX-NLCs were analyzed and the best ratio was determined.
DOI: 10.3109/10717544.2015.1101792
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Characterization of drug loaded NLCs
Cell viability assays in vitro
Particle size and zeta potential
SKOV3 cells or SKOV3/PTX cells were plated at a density of 1 104 cells/well in 96-well plates. Cells were cultured in DMEM medium (supplemented with 10% fetal bovine serum, 100 U/mL of penicillin and 100 mg/mL streptomycin) and maintained at 37 C in a 5% CO2 atmosphere. After the overnight incubation, the medium was replaced with medium containing HA-PTX-NLCs, PTX-NLCs, PTX solution, blank NLCs. Then the cells were incubated at 37 C, 5% CO2 for additional 72 h. The cell viability was measured using the 3 -(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) method according to manufacturer’s instructions.
The measures of mean particle size and polydispersity index (PDI) of different NLCs formulations were performed by dynamic light scattering technique using a Zetasizer (NanoZS90, Malvern Instruments, UK). The zeta potential was measured by the nanoparticles electrophoresis mobility using a U-type tube at 20 C. Drug entrapment efficiency and loading content
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Hyaluronic acid based nanoparticle system
Entrapment efficiency (EE) and loading content (LC) of HAPTX-NLCs and PTX-NLCs were determined were determined using a subtraction method (Zhang et al., 2015b). Briefly, drug-loaded NLCs were dispersed in phosphate buffer solution (2% Tween 80 in PBS, pH 7.4) and vortexed for 3 min to dissolve the free PTX. Then, the dispersion was centrifuged for 0.5 h at 10 000 rpm to separate the free drug from NLCs. After the centrifugation, PTX content in the supernatant was measured by HPLC (Waters2695, Milford, MA). A 20 mL of the solution was injected into an InertsilÕ ODS-3 V analytical column. Mobile phase was consisted of a mixture of acetonitrile and water (50:50, v/v). Flow rate was kept at 1.0 mL/min and system was maintained at 35 C, the detection was carried out at ¼ 227 nm. ðTotal weight of PTX added Weight of free PTXÞ EEð%Þ ¼ Total weight of PTX added 100 ðTotal weight of PTX added Weight of free PTXÞ Total weight of PTX and NLCs 100
LCð%Þ ¼
Drug release study in vitro Paclitaxel (PTX) release from HA-PTX-NLCs, PTX-NLCs and PTX solution was assessed by the dialysis method in PBS 5.5 and 7.4, respectively. Briefly, the NLCs were suspended in 10 mL of the PBS release medium in dialysis membrane (molecular weight cutoff: 10–14 kDa) and transferred to a glass beaker containing 90 ml of PBS (pH 7.4). The samples were incubated at 37 ± 0.5 C with constant agitation of 200 rpm for the release studies. One milliliter of buffer was periodically withdrawn and replaced with equal volume of fresh PBS. The amount of PTX released was determined by HPLC as describe in the above ‘‘EE and LC’’ part. In vitro Stability of HA-PTX-NLCs The stability of HA-PTX-NLCs in 50% fetal bovine serum (FBS) was evaluated by measuring their particle size, PDI and zeta potential after incubation at 37 C for 24 h. At scheduled times (24 h), 1 mL of each sample was diluted with 2 mL THF and the mixture was bath sonicated for 5 min, followed by centrifugation at 10 000r/min for 5 min. PTX content in the supernatant was measured by HPLC method describe in the above ‘‘EE and LC’’ section.
Antitumor efficiency studies in vivo BALB/c mice were subcutaneously inoculating with SKOV3 cells suspended in PBS. When tumor volume reached about 50 mm3, transplanted mice were randomly divided into five groups containing six animals each. HA-PTX-NLCs, PTXNLCs, PTX solution, blank NLCs and normal saline (NS) were injected every six days, respectively. Tumor length and width were measured every 3 days by digital vernier caliper to depict the late stage of the tumor progression. The tumor volume (TV) was calculated by the following equation: TV ¼ Length Width2 =2 The antitumor efficacy of each formulation was evaluated by tumor inhibition rate (TIR), which was calculated using the following equation: Tumor weight of sample group Tumor weight of control group TIRð%Þ ¼ Tumor weight of control group 100 The body weight changes of different groups were also recorded. Biodistribution investigation BALB/c mice were treated the same way mentioned in the ‘‘Antitumor efficiency studies in vivo’’ section. At predetermined time intervals, mice were sacrificed and the tumor, heart, liver, spleen, lung and kidney of mice were collected and stored. To determine the amount of PTX in each tissue, tissues were first weighed and homogenized with physiological saline. Then, PTX was extracted with acetonitrile and methanol (1:1, v/v) and centrifuged (15 000 rpm, 10 min). The supernatant was collected, and the amount of PTX was determined by HPLC as describe in the above ‘‘EE and LC’’ section. Statistical analysis All studies were repeated three times and all measurements were carried out in triplicate. Results were reported as means ± SD (SD ¼ standard deviation). Statistical significance was preformed by two-tailed Student’s t-test at p50.05.
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Results and discussion
Table 1. Characterization of different vectors.
Characterization of drug loaded NLCs
Samples
Blank NLCs
PTX-NLCs
HA-PTX-NLCs
Lipid nanoparticles can take advantage of the enhanced permeation and retention (EPR) effect, and have been shown to overcome MDR mechanisms by directing endocytosis-mediated cellular internalization of drug and/or interacting directly with efflux pumps (Hadinoto et al., 2013; Nandini et al., 2015). Size and distribution are important factors for the nanocarriers, which can influence the efficiency of the drug loaded nanoparticles (Jin et al., 2007; Nandini et al., 2015). Particle size around 100 nm could decreases uptake by the liver, prolongs circulation time in the blood and improves bioavailability. The mean particle size of HA-PTX-NLCs, PTX-NLCs, blank NLCs was 114.3, 96.1 and 93.6 nm, respectively (Table 1). No significant change in diameter was found from blank NLC and PTX-NLCs; however, the size of HA-PTX-NLCs was larger due to the coating of the HA ligands. The zeta potential is a key factor for the stability of nanosystems (Xu et al., 2009). The positive charge of the NLCs and also facilitate the delivery of drug to the negatively charged cancer cells. The zeta potential of HA-PTX-NLCs, PTXNLCs and blank NLCs was +11.5, +29.6 and +17.9 mV, respectively. Higher zeta potential of PTX-NLCs than blank NLCs may contribute to the positively charged PTX. The zeta potential of HA-PTX-NLCs were the lowest, due to the negatively charged HA neutralized the charge of the cationic NLCs. The EE of HA-PTX-NLCs, PTX-NLCs was over 80%, and the LC was 5.6% for PTX-NLCs and 3.2% for HA-PTX-NLCs.
Particle size (nm) Size distribution Zeta potential (mV) EE (%) LC (%)
93.6 ± 3.1 0.10 ± 0.02 +17.9 ± 2.3 N/A N/A
96.1 ± 3.4 0.13 ± 0.02 +29.6 ± 3.4 83.1 ± 3.2 5.6 ± 0.6
114.3 ± 4.6 0.16 ± 0.03 +11.5 ± 1.7 81.9 ± 2.8 3.2 ± 0.5
Table 2. Optimization of HA-PTX-NLCs. HA-DSPE to PTX-NLCs ratio (w/w) 1:3 1:2 1:1 2:1 3:1
Zeta potential (mV)
Particle size (nm)
EE (%)
+24.3 ± 4.1 +18.6 ± 2.8 +11.7 ± 1.9 +10.1 ± 2.6 +9.9 ± 2.2
99.7 ± 3.4 105.3 ± 3.7 113.4 ± 4.6 237.6 ± 45.2 339.9 ± 64.7
82.3 ± 2.6 81.7 ± 2.7 82.4 ± 2.4 61.2 ± 6.4 38.3 ± 8.2
Optimization of HA-PTX-NLCs Hyaluronic acid (HA) is an anionic glycosaminoglycan. HA has an important biological role in the cell adhesion, migration, invasion, proliferation, differentiation and angiogenesis by binding to cell specific receptors like glycoprotein CD44, who was considered to be associated with drug resistance and is an unfavorable prognosis or survival marker in cancers including ovarian cancer (Ghosh et al., 2012; Saadat et al., 2014). To optimize the best HA-PTXNLCs formulation, different ratio of HA-DSPE to PTXNLCs (1:3, 1:2, 1:1, 2:1, 3:1, w/w) were used, and the property of the resulting HA-PTX-NLCs were analyzed (Table 2). The property of the HA-PTX-NLCs would be changed as follows due to the adding of HA ligands: (1) the presence of anionic HA could neutralize the positive potential of the cationic NLCs, cause the change of potential. (2) The HA-DSPE could be the shell of the HAPTX-NLCs, thus increased the particle size. It is worth noting that the exclusive using of HA ligands may cause the leap of the size and the wide size distribution. (3) The EE should be maintained at a high level; significantly decrease of EE may be the evidence of the instable of the NLCs system. We can tell from Table 2 that the ratio at 1:1 of HA-DSPE to PTX-NLCs was best for all the parameters of HA-PTXNLCs, including stable zeta potential and EE, suitable particle size and narrow size distribution.
Figure 2. PTX release from HA-PTX-NLCs, PTX-NLCs, and PTX solution in PBS 7.4.
Drug release study in vitro Paclitaxel (PTX) release from HA-PTX-NLCs and PTXNLCs was assessed in PBS 7.4 (Figure 2) and 5.5 (Figure 3), respectively. Figures 2 and 3 shown that: (1) PTX release from both HA-PTX-NLCs and PTX-NLCs were faster in the acidic (pH 5.5) than neutral environment (pH 7.4), this could be the evidence that the drug would release more easily in acidic tumor tissue; (2) PTX release from HA-PTX-NLCs were slower than PTX-NLCs, this may due the HA appearing on the surface that delayed the release of drugs. The release profile shows that HA-PTX-NLCs formulation has the capacity to release PTX at a sustained rate, let the anticancer drug to maintain continuous efficiency. In vitro Stability of HA-PTX-NLCs Considering the intravenous administration usage of the drug loaded NLCs, the stability of HA-PTX-NLCs and PTX-NLCs
Hyaluronic acid based nanoparticle system
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Figure 5. Cell viability of HA-PTX-NLCs, PTX-NLCs and PTX solution in SKOV3/PTX cells. Figure 3. PTX release from HA-PTX-NLCs, PTX-NLCs, and PTX solution in PBS 5.0.
Table 3. Stability of HA-PTX-NLCs in 50% FBS.
Samples HA-PTX-NLCs PTX-NLCs
Table 4. IC50 of HA-PTX-NLCs, PTX-NLCs, PTX solution evaluated in SKOV3 cells and SKOV3/PTX cells. Samples
Particle size (nm)
Size distribution
Zeta potential (mV)
118.7 ± 5.9 101.4 ± 4.2
0.19 ± 0.05 0.15 ± 0.03
17.6 ± 2.4 8.6 ± 0.9
Figure 4. Cell viability of HA-PTX-NLCs, PTX-NLCs, and PTX solution in SKOV3 cells.
in serum was investigated to check for aggregation in the presence of proteins. The particle size, size distribution, and zeta potential are described in Table 3. The size and distribution of HA-PTX-NLCs and PTX-NLCs were slightly increased after incubation with serum, but data shown no significant changes (p40.05). The zeta potential of the NLCs in the presence of serum was inversed from positive
SKOV3 cells (mM) SKOV3/PTX cells (mM)
HA-PTX-NLCs
PTX-NLCs
Free PTX
0.011 ± 0.003 0.019 ± 0.004
0.024 ± 0.006 0.045 ± 0.008
0.216 ± 0.031 23.156 ± 2.351
Figure 6. The tumor growth inhibition effect of different groups.
Table 5. Tumor inhibition rates of samples in ovarian cancer bearing mice. Samples
HA-PTX-NLCs
PTX-NLCs
Free PTX
TIR (%)
85.0 ± 4.1
72.6 ± 3.3
25.1 ± 1.9
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to negative. This phenomenon may due to the negatively charged serum that changed the surface charge of the positive NLCs (Le Broc-Ryckewaert et al., 2013). The relatively stable characteristic illustrated that HA-PTX-NLCs and PTX-NLCs were stable in serum and would not aggregate after intravenous administration.
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Cell viability assays in vitro
Figure 7. Body weight changes of different groups.
Figure 8. Mean tissue concentrations of PTX after injection of PTX solution.
Figure 9. Mean tissue concentrations of PTX after injection of HA-PTX-NLCs.
In vitro SKOV3 and SKOV3/PTX cells cytotoxicity was evaluated by MTT assay to evaluate the activity of drug loaded NLCs against ovarian cancer cells. Figures 4 and 5 showed the HA-PTX-NLCs, PTX-NLCs and PTX solution reduced cell viability in a concentration-dependent manner. At all the studied PTX concentrations, the cytotoxicity of HAPTX-NLCs was higher than PTX-NLCs (p50.05), tumor cell inhibition effect of drug loaded NLCs was better than free PTX solution (p50.05). The IC50 values of HA-PTXNLCs, PTX-NLCs and PTX solution were presented in Table 4. PTX-NLCs significantly decreased the IC50 value 10 times compared to PTX solution (p50.05), implying that NLCs formulations show higher cytotoxicity against ovarian cancer cells. HA-PTX-NLCs decreased the IC50 value two times dose advantage on PTX-NLCs (p50.05). The enhanced efficacy of PTX loaded NLCs against ovarian tumor cells may be explained mechanism that the enhanced intracellular drug accumulation by ligand-receptor recognition, and by nanosized carrier uptake (Liu et al., 2011).
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Antitumor efficiency studies in vivo
References
In vivo antitumor efficiency of NLCs was evaluated in mice bearing ovarian cancer xenograft. The tumor growth inhibition effect was shown in Figure 6 in terms of the tumor volume changes. The TIR of different groups were illustrated in Table 5. HA-PTX-NLCs group exhibited the highest TIR (85%), followed by PTX-NLCs (73%) and PTX solution (25%). The curves of tumor volume and TIR showed that: (1) obvious tumor regression was observed in mice treated with HA-PTX-NLCs, PTX-NLCs, PTX solution, while no antitumor effect was observed in the blank NLCs and NS groups; (2) tumor growth was more significantly inhibited by drug loaded NLCs groups (HAPTX-NLCs and PTX-NLCs) than the PTX solution (p50.05); (3) HA-PTX-NLCs showed better anti-tumor efficacy than PTX-NLCs (p50.05), the most obviously tumor regressions were observed in the HA-PTX-NLCs group, the tumor growth was prominently inhibited than any other group (p50.05). Body weight changes were illustrated in Figure 7. Obviously decrease in mean body weights were found in blank NLCs and NS group, with the reduced foods intake, moving inactively. However, slightly increase of body weights were found in HA-PTX-NLCs and PTX-NLCs, the foods intake and movement is normal. These results may suggest the less systemic toxic side effect of the NLCs formulations for the in vivo cancer treatment.
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Biodistribution investigation Figure 8 showed that the concentration of PTX was higher in the heart and kidney for the PTX solution group, which may explain the adverse side effects. However, the concentrations of PTX in the heart and kidney were lower for the HA-PTX-NLCs group (Figure 9), which was expected to reduce the side effects. The concentration of PTX for the HA-PTX-NLCs group was higher in the tumor and liver compared with the drugs solution group, which may be due to the size of NLCs and the EPR effect of nanocarriers in the tumor. Higher concentrations of HAPTX-NLCs were found in tumor tissue during 12–48 h after intravenous injection, shown the sustained release behavior of the HA-PTX-NLCs.
Conclusions For ovarian cancer chemotherapy, SKOV3 cells and mice bearing ovarian cancer model were used to evaluate the efficiency of HA based, PTX loaded NLCs system. The results suggest that this drug delivery system could be stable in serum, achieve targeted delivery of drug, reduce the systemic toxicity and reach the outstanding antitumor efficiency. These findings strongly support the superiority of HA based nano-system for the delivery of drugs and for the targeted treatment of ovarian cancer.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
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