http://informahealthcare.com/phd ISSN: 1083-7450 (print), 1097-9867 (electronic) Pharm Dev Technol, Early Online: 1–9 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/10837450.2014.965323
RESEARCH ARTICLE
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Pluronic F127 polymeric micelles for co-delivery of paclitaxel and lapatinib against metastatic breast cancer: preparation, optimization and in vitro evaluation Pooya Dehghan Kelishady, Ebrahim Saadat, Fatemeh Ravar, Hamid Akbari, and Farid Dorkoosh Department of Pharmaceutics, Faculty of Pharmacy, Tehran University of Medical Science, Tehran, Iran
Abstract
Keywords
The aim of this study was to develop and characterize the paclitaxel (PTX)-lapatinib (LPT) loaded micelles for simultaneous delivery against metastatic breast cancer. Efflux pumpmediated drug resistance influences the efficacy of chemotherapeutic regimens. However, in the newly developed delivery system, LPT was selected to act as chemosensetizer. LPT increases the intracellular level of PTX by inhibition of efflux pumps. Pluronic F127 was selected for the preparation of the micelles, and its critical micelle concentration was determined to be 0.012 mg/ml. D-optimal design was used to analyze the impact of different experimental parameters on PTX and LPT encapsulation ratio. PTX encapsulation ratio was optimized at 68.3%, while LPT encapsulation ratio found to be 70.1%. Transmission electron microscope analyses demonstrate that micelles possess a good core–shell structure without any sharp edge. Laser scattering method results indicated that size of the optimized micelles is 64.81 nm with acceptable polydispersity index (0.309). In vitro release studies showed a sustain release pattern. PTX–LPT-loaded micelles suppressed the proliferation of resistant T-47D cell line (IC50 ¼ 0.6 ± 0.1 mg/ml) compared to binary mixture of PTX and LPT (IC50 ¼ 6.7 ± 1.2 mg/ml). Therefore, it is concluded that the developed formulation might increase the therapeutic efficacy in drug resistant metastatic breast cancer.
D-optimal design, drug resistance, efflux pump, lapatinib, paclitaxel, polymeric micelle
Introduction Paclitaxel (PTX) is a common chemotherapeutic agent which is widely applied for the treatment of different types of solid tumors1. PTX suppresses cancer cells proliferation by disturbing microtubule network during the cell division1. PTX also triggers apoptosis (programmed cell death) of cancerous cells by G2/M cell-cycle arrest2,3. However, PTX restricts the proliferation of normal cells which leads to severe side effects decreasing patients compliance during chemotherapy period4. Unfortunately, poor water solubility of PTX (0.3 mg/ml) is a major obstacle upon application of PTX5. In the commercial formulation of PTX, TaxolÕ , the solubility is enhanced by Cremophor ELÕ as a solubilizing agent. However, several studies reported serious side effects of Cremophor ELÕ including hypersensitivity, nephrotoxicity and neurotoxicity6. There are two main novel formulations for PTX, AbrexaneÕ and Genexol-PMÕ , which have been developed as alternatives for TaxolÕ in chemotherapeutic regimens. AbrexaneÕ is an albumin bound PTX nanoparticle which has fewer and limited side effects compared to TaxolÕ 7. However, the high-cost level of AbrexaneÕ restricts its use in clinic8. Genexol-PMÕ (Samyang Co., Seoul, Korea) is the polymeric micelle form of PTX developed by PEG-PLA co-polymer9.
Address for correspondence: Dr. Farid Dorkoosh, Department of Pharmaceutics, Faculty of Pharmacy, Tehran University of Medical Science, Tehran, Iran. E-mail: [email protected]
History Received 29 April 2014 Revised 8 August 2014 Accepted 6 September 2014 Published online 29 September 2014
Several drug delivery systems (DDS) have been designed to overcome PTX solubility problem as well as minimizing PTX side effects on normal cells. DDS including polymeric micelles10,11, liposomes12,13, nanospheres14, nanoparticles15, polymer conjugates16 and dendritic polymers17,18 are all designed for delivery of PTX. In these series of studies, the aqueous solubility of PTX is enhanced to some extent but the resistance of cancerous cells is still a challenging issue. Multidrug resistance (MDR) is one of the main complications which could be developed by cancerous cells. Efflux-mediated drug resistance is mainly provoked by ATP-binding cassette (ABC) family including P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP)19. Efflux pumps decrease the intracellular level of the cytotoxic agents and, as a result, restrict the therapeutic efficacy. A strategy that has been investigated to overcome the efflux-mediated drug resistance is to co-administer a P-gp inhibitor along with the anticancer drug. However, P-gp inhibitors can aggravate the side effects. Therefore, among the alternatives for efflux pump inhibitors, the benefits of chemotherapeutic agents which reduce cancer cells proliferation and inhibit the efflux pumps simultaneously are two-fold. Lapatinib (LPT) is a dual tyrosine kinase inhibitor which targets both human epidermal growth factor receptors (EGFR) and HER2 receptors20. Clinical data demonstrate that LPT monotherapy would be helpful in HER2 positive breast cancer21. In metastatic breast cancer, PTX and LPT combination therapy is one of the main therapeutic regimens clinically
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applied22,23. Recently, some reports demonstrated that tyrosine kinase inhibitors such as LPT act as an efflux pump inhibitor besides of their cytotoxic properties. LPT binds to the internal domain of efflux pump and inhibits the function of transporter therefore sensitizes the MDR cancerous cells to chemotherapeutic agents such as PTX24–26. Since LPT increases intracellular level of PTX by inhibiting efflux pumps, designing a DDS for co-delivery of PTX and LPT would be a good choice (Figure 1). DDS such as polyelectrolyte nanocapsules27 and lipopolymer micelles28 have been developed to deliver PTX and LPT together for treatment of ovarian and prostate cancer, respectively. PTX–LPT dual delivery might be efficient in treatment of metastatic breast cancer since LPT combined with PTX offers a significant and clinically meaningful survival advantage over PTX alone in patients with HER2-positive metastatic breast cancer23. Polymeric micelles have been widely used to enhance the solubility of hydrophobic drugs. Core–shell structure of the micelles enables them to encapsulate the hydrophobic agents, while the hydrophillic shell enhances the stability of the particle. Tumor tissues possess a leaky vascularized network which allows large polymeric complexes to easily overpass the endothelial layer and accumulate in the tumor cells29. This phenomenon is called enhanced permeability and retention (EPR) effect. EPR allows the polymeric micelles to target passively the tumor cells. Protection from inactivation in biological media and long circulation period which enhance the pharmacokinetics of PTX and LPT are the other advantages of polymeric micelles30,31. Pluronic di-block polymers are made of hydrophilic poly ethylene oxide (PEO) and hydrophobic poly propylene oxide (PPO) arranged in di-block structure. Amphiphilic nature of this polymer makes it an appropriate choice for preparation of polymeric micelles. Pluronic polymers induce ATP depletion and consequently cause P-gp inhibition in MDR cells32. Pluronic F127 (PEO100–PPO69–PEO100) is commonly used to improve the stability and encapsulation efficiency of micelles. F127 was selected in order to prepare the micelles due to its biocompatibility and its approval by FDA. The purpose of this study was the development of F127 polymeric micelles encapsulating PTX and LPT as chemotherapeutic agents. Critical micelle concentration (CMC) of Pluronic F127 was determined for assuring that micelles would maintain their form in diluted conditions such as biological media. Encapsulation ratio (EN%) for PTX and LPT was modeled and optimized using response surface methodology approach. Optimized formulation was selected according to PTX and LPT EN%. Morphology of PTX–LPT-loaded micelles was studied by Transmission Electron Microscopy (TEM). Laser scattering method revealed the characteristics of micelles such as shape, form and size. In vitro release test defined the release Figure 1. Aim of design of dual delivery system for PTX and LPT.
Pharm Dev Technol, Early Online: 1–9
pattern of PTX and LPT from polymeric micelles. Finally, PTX–LPT-loaded micelle’s cytotoxicity was evaluated by MTT assay on resistant T-47D cell line as breast cancer model. In vitro cytotoxicity assay promoted us to expand our studies to in vivo tests in future studies which can determine efficacy of DDS in animal models.
Materials and methods Materials LPT ditosylate (99.9% purity) was purchased from Beijing Mesochem Technology Co. (Beijing, China) and used without further purification. PTX (99% purity) was obtained from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Pluronic F127 was purchased from Gilson Co. (Villiers-le-Bel, France). T-47D cell line was obtained from National Cell Bank of Iran (NCBI), Pastor Institute of Iran. 3-(4,5-Dimethyl-thiazol-2yl)-2,5-diphenyl-tetrazolium bromide (MTT) was purchased from Sigma (St. Louis, MO). Penicillin–streptomycin, RPMI 1640, fetal bovine serum (FBS) and 0.25% (w/v) trypsin–0.03% (w/v) EDTA solution were purchased from Gibco BRL (Gaithersburg, MD). All other solvents and reagents were either analytical or chromatographic grade and used as received. Critical micelle concentration determination In order to determine critical micelle concentration (CMC) of Pluronic F127 in deionized (DI) water, pyrene probe method was applied33. Briefly, pyrene method determines the CMC by analyzing pyrene content incorporating into the micelles. In concentrations around the CMC value, there is a sharp decrease in pyrene spectroflourimetry signals due to its incorporation to the micelles. 5 mg of pyrene was dissolved in acetone and serially diluted to 6.0 106 M using A grade volumetric flasks and A grade volumetric pipettes. 1 ml aliquot of the pyrene solution was added into 10 ml volumetric flasks. Acetone content of each flask was evaporated completely under mild heating. Each flask was filled up with 10 ml of F127 dispersions in DI water (concentrations ranging over 5 104 to 5 mg/ml). The final concentration of pyrene was controlled at 6.0 107 M. The solutions were sonicated for 30 min and then stirred at 700 rpm overnight in the room temperature. The spectroflourimetry analyses were performed by a spectroflourimeter (Shimizu, Tokyo, Japan). Pyrene was excited at 334 nm, and its emission was recorded at 373 and 384 nm, which corresponds to the first and third vibrational peaks, respectively. Excitation and emission slits set at 8 and 2 nm, respectively. The intensity ratio of the first peak (I1, 373 nm) to the third peak (I3, 384 nm) was plotted against the polymer concentration and analyzed for the calculation of CMC.
PTX–LPT-loaded pluronic polymeric micelles
DOI: 10.3109/10837450.2014.965323
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Preparation of PTX–LPT-loaded micelles PTX–LPT-loaded micelles were prepared by thin-film hydration method34. Various defined amounts of PTX, LPT and F127 were dissolved in 10 ml acetonitrile using a round bottom flask. Acetonitrile was evaporated by rotary evaporator at about 50 C. Residual acetonitrile content was evaporated under the vacuum condition overnight in order to reassure that organic solvent was eliminated from final formulation completely. In the following day, thin film was hydrated by 10 ml of distilled water at 60 C and stirred for 30 min to prepare the micellar solution. The solution was filtrated through 0.22 mm cellulose acetate (CA) filter to eliminate unincorporated drug aggregates from the final micellar solution. Obtained micellar solution was lyophilized and stored in 5 C for further drug content analysis. Drug content analyses and HPLC procedure PTX and LPT contents were analyzed by HPLC system (Agilent Technologies model 1260 Infinity) which was equipped with an autosampler system (Agilent Technologies, Santa Clara, CA) fitted with a 25-ml sample loop and G1315D diode array detector (Agilent Technologies, Santa Clara, CA). A reversedphase column (C18 MZ-Analytical column, 5 mm, 150 4.6 mm, OSD-3) which was protected by pre-column (5 mm, 4.0 4.6 mm, OSD-3) was used at room temperature. Chromatographic data was analyzed by Agilent Chemostation for LC system software (B.04.02). The mobile phase consisted of acetonitrile and deionized water (70/30, V/V). Mobile phase was freshly prepared for each run and filtered through PTFE filter (0.45 mm). Injection volume was set at 20 ml, and flow rate was adjusted at 1.5 ml/min. Detection wave length was monitored at 227 nm. The lyophilized PTX–LPT micelles were hydrated by DI water. 1 ml of micellar solution was mixed with the equal volume of acetonitrile and sonicated in bath sonicator for 10 min in order to disturb the micelle structure. 20 ml of extracted solution was injected into the HPLC to analyze PTX and LPT incorporated into the micelles. Typical retention times for PTX and LPT were found out to be 2 and 4 min, respectively. The concentrations of PTX and LPT were determined by comparing the peak areas with the standard calibration curve.
Experimental design D-optimal design D-optimal method was conducted to figure out the effect of major parameters on the micelle’s characteristics in a few experimental runs35,36. Major micelle’s characteristics which are defined as responses are mainly depended on different experimental parameters in various degrees of interaction. D-optimal design was used to analyze the impact of three different major parameters, including feeding PTX (X1), feeding LPT (X2) and feeding F127 (X3). Both PTX and LPT feeds were ranged over 1 to 5 mg, while the polymer feed was selected to be 50–150 mg. Former studies on similar formulations and also results of some preliminary experiments were used in order to select the major parameters and their levels in the experimental design37,38. PTX encapsulation ratio (PTX EN%, Y1) and LPT encapsulation ratio (LPT EN%, Y2) were calculated, and their surface response was analyzed because they were generally regarded as the major characteristics of developed polymeric micelles. Encapsulation ratios and also drug-loading coefficients (DL%) were calculated using the following equations:
ER% ¼
DL% ¼
3
weight of the drug in micelles 100 weight of the feeding drug
weight of the drug in micelles 100 weight of the feeding polymer and drug
Experimental designs, analysis and optimization were performed using Design expertÕ (version 7.0.0, stat-Ease, Inc., Minneapolis, MN). Eighteen experimental runs were conducted using D-optimal design and two responses were obtained for further assessments. D-optimal design, as a technique, assists the selection of the model which can precisely and accurately define parameter–response relationship. According to the previous studies, quadratic and 2FI models mainly define this relationship and are introduced as follows: Yn ¼ b0 þb1 X1 þb2 X2 þb3 X3 þ . . . þb12 X1 X2 þb13 X X3 þ . . . þb11 X12 þb22 X22 þb33 X32 ðQuadratic modelÞ Yn ¼ b0 þb1 X1 þb2 X2 þb3 X3 þ . . . þb12 X1 X2 þb13 X X3 ð2FI modelÞ In both 2FI and quadratic models, X1 to X3 represent individual effect of parameter, whereas X1X2 and other combination of factors show the binary interactions of parameters on each other. X 21 to X 23 demonstrate second-order interaction of parameters on the response surface in the quadratic model. Significancy of the models and their coefficients were investigated by Analysis of Variance (ANOVA). Optimization procedure Optimization procedure was performed using numerical optimization by Design expertÕ (version 7.0.0, stat-Ease, Inc., Minneapolis, MN). In brief, our goal was to maximize the encapsulation ratio for PTX and LPT. In practice, it is unlikely to maximize all parameters simultaneously. Therefore, numerical optimization method was performed which is based upon utilization of desirability functions. Each single response has its own desirability (d1 to d2). Individual desirability can be used to calculate the overall desirability by below equation in which D represents overall desirability and d1 to d2 is corresponding to individual desirabilities: D ¼ðd1 d2 Þ1=2 Overall desirability function represents the overall optimization state. Design expertÕ nominates the best solutions; we selected the best formulation according to the overall desirability function and also with respect to individual desirabilities. Particles size measurements and morphology study Size, polydispersity index (PI) and zeta potential measurements were conducted by Malvern zetasizer (Malvern Instruments Ltd, Malvern, UK). The analyses were performed with 5mWHe–Ne laser (632.8 nm) at a scattering angle of 90 at 25 C. Lyophilized powder was hydrated with 10 ml DI water. For appropriate size analysis and avoiding multi-scattering phenomena, obtained micellar solution was diluted in 10 ml of DI water. Afterwards, samples were vortexed well and placed in quartz cuvette for size measurements. Morphology studies of PTX–LPT-loaded micelles
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were carried out by Transmission Electron Microscope (TEM). Prior to visualization, samples were diluted and fixed on a grid for microscopic analyses.
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In vitro release of PTX and LPT from drug-loaded micelles In vitro release of the optimized PTX–LPT-loaded micelles was performed in an aqueous solution using dialysis method in sink condition39. 1 ml PTX–LPT-loaded micelle solution was added to a dialysis bag [molecular weight cutoff (MWCO) ¼ 12 000 Da]. Dialysis bag was end-sealed completely and fully immersed in 100 ml PBS (pH 7.4) containing PEG 400, 10% (V/V). PEG 400 added to enhance the solubility of PTX and maintain the sink condition5,40. Release medium was maintained at 37 ± 0.5 C and stirred at 100 rpm for 48 h. At predetermined time intervals, 1 ml aliquot was withdrawn for further analyses. Instantly after sampling, entire release medium was substituted by fresh medium for avoiding PTX and LPT accumulation in medium and maintaining the sink condition. Samples were analyzed by RP-HPLC method discussed earlier, and the amount of released PTX and LPT was determined. Finally, the cumulative release of PTX and LPT was plotted against time to evaluate the in vitro release profiles. In vitro cytotoxicity assay Resistant T-747D cell line which is derived from mammary glands’ metastatic sites was selected as a model for metastatic breast cancer. T-47D was cultured at 37 C and 5% CO2. Culture medium consisted of RPMI 1640 medium supplemented by 10% FBS, 100 IU/ml penicillin and 100 mg/ml streptomycin sulfate. Cells were seeded at the density of 5 103 cells per well in 96-well plates. After 24 h of incubation at 37 C with 5% CO2, the growth medium was replaced by 200 ml medium containing Pluronic F127, binary mixture of PTX and LPT in DMSO and PTX–LPT-loaded micelles in concentrations ranging from 0.001 to 1000 mg/ml. After 72 h incubation, cell survival was measured using tetrazolium salt (MTT) assay. 180 ml of fresh growth medium and 20 ml of MTT (5 mg/ml) solution were added to each well. The plate was incubated for an additional 4 h, and then 200 ml of DMSO was added to each well to dissolve any purple formazan crystals formed. The plates were vigorously shaken before measuring the relative color intensity. The absorbance at 570 nm of each well was measured by a microplate reader (BioTek ELx 800, USA). Each assay was repeated in triplicates.
Results and discussion CMC determination Critical micelle concentration is an important factor which reveals the stability of micelle both in vivo and in vitro. Micelles may disassemble in diluted media and it is an important issue especially in DDS. When the micelles are administered intravenously, they are being diluted several times in blood stream. If the CMC value of micelle is low enough, it means that micelle structure remains intact even after several dilutions. On the other hand, micelles with high-CMC values may be disassembled and immaturely release their payload which can cause toxicity and other side effects. Pyrene method is a simple procedure for CMC determination. Pyrene incorporates into the micelles due to its hydrophobe nature, while the unincorporated pyrene remains in the aqueous medium and demonstrate florescence properties of pyrene. Ratio of the first vibrational peak (I1, 373 nm) to the third vibrational peak (I3, 384 nm) indicates the unincorporated pyrene content. Around CMC value because of initiation of micelle forming process, pyrene starts to incorporate into the micelles and consequently a rapid I1/I3 decrease is observed.
Figure 2. Plot of I1/I3 versus concentration of F127 in DI water.
Plotting I1/I3 against logarithm of polymer concentration shows two different bunches of points in the plot: high I1/I3 group and low I1/I3 group (Figure 2). Two regression lines were plotted by these two groups of points. The interaction point of these two lines indicates the CMC value. CMC value found to be 0.012 mg/ml which is relevant with previously measured CMC value by iodine UV spectrometry method37. Measured CMC value indicates that F127 polymer possesses a good micelle forming capability compared to the other pluronic polymers which make it suitable for preparation of the polymeric micelles37,41. Design of experiment D-optimal design Eighteen experimental runs (Table 1) were conducted by D-optimal design to analyze and model the impact of feeding PTX (X1), feeding LPT (X2) and feeding F1127 (X3) on the PTX encapsulation ratio (PTX EN%, Y1) and LPT encapsulation ratio (LPT EN%, Y2). PTX encapsulation ratio has ranged from 2.82 to 88.90%. This wide response range demonstrates that PTX encapsulation ratio is strongly depended on experimental parameters. Drug loading coefficient for PTX also has ranged from 0.22 to 1.43%. Statistical analysis shows that PTX encapsulation ratio fitted into a quadratic model [Equation (1)] with significant p value (p value50.05) and also insignificant lack of fit (p value40.05) (Table 2). PTX EN% ¼ 59:83114 43:00824 PTX þ 44:30701 LPT 0:29956 polymer þ 5:0918 PTX2 7:23246 LPT2 þ 2:53353 3
10 polymer2 þ 0:29835 PTX LPT
ð1Þ
6:17912 103 PTX polymer þ 9:99386 103 LPT polymer F127 Polymer feed has increased EN% for both PTX and LPT since it increases the amount of polymer available for encapsulating the hydrophobe PTX and LPT. A significant (p value50.05) first-order impact of polymer is suggested for PTX EN% and LPT EN%. According to the literature, similar result was reported for encapsulation of vincristine into PLGA nanoparticles42,43. First- and second-order impact of PTX feed on PTX EN% was
PTX–LPT-loaded pluronic polymeric micelles
DOI: 10.3109/10837450.2014.965323
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Table 1. D-optimal experimental runs.
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Experimental parameters (mg) Std
Run
PTX feed
LPT feed
Polymer feed
PTX encapsulation
LPT encapsulation
LPT loading
PTX loading
11 12 16 2 15 13 8 7 5 9 4 18 10 14 3 17 6 1
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
4.00 1.00 1.00 1.00 1.00 5.00 1.00 3.00 1.00 5.00 5.00 1.00 3.00 5.00 5.00 5.00 3.00 1.00
3.00 5.00 1.00 1.00 5.00 5.00 3.00 3.00 1.00 1.00 1.00 1.00 5.00 5.00 5.00 1.00 1.00 5.00
75.00 50.00 50.00 150.00 150.00 50.00 100.00 150.00 50.00 50.00 150.00 150.00 50.00 150.00 100.00 150.00 100.00 150.00
21.14 62.52 53.72 70.45 88.9 16.8 85.86 75.19 50.01 2.52 24.6 69.11 6.47 27.45 26.06 12.44 11.95 75.11
83.95 16.68 45.81 53.14 72.75 48.24 51.81 60.54 46.14 63.89 75 51.95 11.33 78.72 69.42 52.55 77.53 75.68
3.07 1.49 0.88 0.35 2.33 4.03 1.49 1.16 0.88 1.14 0.48 0.34 0.97 2.46 3.4 0.33 0.73 2.42
1.03 1.12 1.03 0.46 0.56 1.4 0.82 1.43 0.96 0.22 0.79 0.45 0.33 0.86 1.18 0.4 0.34 0.48
Table 2. D-optimal analysis of PTX EN%.
Source Model X1 X2 X3 X21 X22 X23 X1X2 X1X3 X2X3 Residual Lack of fit Pure error Cor total
Response (%)
Table 3. D-optimal analysis of LPT EN%.
Sum of SQUARES
Degree of freedom (df)
Mean squares
14100.53 7605.20 447.68 1531.02 873.23 1427.73 84.45 17.31 4.26 12.14 791.34 614.54 176.79 14891.87
9 1 1 1 1 1 1 1 1 1 8 4 4 17
1566.73 7605.20 447.68 1531.02 873.23 1427.73 84.45 17.31 4.26 12.14 98.92 153.64 44.20
F value
p value*
Source
Sum of squares
Degree of freedom (df)
Mean squares
15.84 76.88 4.53 15.48 8.83 14.43 0.85 0.18 0.043 0.12
0.0003 50.0001 0.0661 0.0043 0.0178 0.0052 0.3825 0.6867 0.8407 0.7351
Model X1 X2 X3 X1X2 X1X3 X2X3 Residual Lack of fit Pure error Cor total
4983.84 1385.41 203.71 1738.12 5.12 301.83 2074.64 1884.82 1627.76 257.06 6868.66
6 1 1 1 1 1 1 11 7 4 17
830.64 1385.41 203.71 1738.12 5.12 301.83 2074.64 171.35 232.54 64.26
3.48
0.1274
*Significant at 0.05 level.
significant (p value50.5). Higher PTX EN% is achieved for lower PTX feeding (Figure 3). There is a limited capacity in the core part of pluronic micelle which would be saturated by defined amount of PTX. Therefore, by increasing the total amount of PTX feed, the micelle core would reach to the saturation which leads to decrease in EN% of PTX accordingly. As it is reported, hydrophobic interactions among PTX molecules are greater than PTX and Pluronics44. Therefore, due to the strong hydrophobic interaction among PTX molecules and low solubility of PTX in water, unincorporated PTX aggregates would eventually form in the medium. These drug aggregates would limit available free PTX for encapsulating into the micelles and consequently reduce PTX EN%. PTX EN% is being maximized in medium concentrations of LPT ranging from 2 to 4 mg. The model suggests a significant second-order relation (p-value50.05). It is clear that large amounts of LPT would form unincorporated LPT aggregates that may adsorb free PTX in the medium. In analogous study focusing on PTX and tariquidar co-delivery systems, an increase in amount of tariquidar has resulted a reduction in PTX EN%45.
F value
p value*
4.85 8.09 1.19 10.14 0.030 1.76 12.11
0.0117 0.0160 0.2989 0.0087 0.8659 0.2113 0.0052
3.62
0.1157
*Significant at 0.05 level.
On the other hand, LPT EN% has decreased in small LPT feeds. The formation of drug aggregates in small feeding amounts of LPT is negligible; consequently, LPT freely entrapped and fill the core of micelles before PTX due to its more hydrophobe nature. Such a decrease of EN% is also reported in doxorubicinelacridar nanoparticles46. In conclusion, LPT feeds higher than 4 mg and lower than 2 mg would limit PTX EN%. LPT EN% has ranged from 11.83 to 83.95% which is apparently a wide range. Drug loading coefficient for LPT has also ranged from 0.33 to 4.03%. LPT EN% is analyzed by a significant 2FI model (p-value50.05) with insignificant lack of fit (p-value40.05) (Table 3) as follows: LPT EN% ¼ 49:87694 þ 9:80502 PTX 15:25792 LPT 2:51603 103 polymer þ 0:16007 PTX LPT 0:051864 PTX polymer þ 0:12885 LPT
ð2Þ
polymer LPT EN% is inversely related to LPT feeding amount (Figure 3) due to the limited encapsulation efficency of polymer and also acceleration of drug aggregates formation in
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Figure 3. Reponse–parameter relationship (a) PTX EN% (b) LPT EN%.
concentrated intial feeds. The increase of the entrapped amount of LPT was not in proportion to the increase of the initial drug content, thus the entrapment efficiency was decreased42. Mean encapsulation ratio for LPT (57.51%) is slightly greater than PTX (43.35%) and it is less depended on the primary feed, and the model shows only a significant first order relation (p-value50.05). Because of more hyrophobic nature of LPT compared to PTX, it seems that LPT has more conviniently entered into the micelle core. Interestingly, PTX feed raises LPT EN%. As mentioned before, PTX EN% is limited by increasing PTX feed. It seems reasonable to state that by decreasing PTX EN%, significant portion of micelle core is being occupied by LPT.
Table 4. The observed and predicted response values for the optimized formulation. Parameter
Optimized level
X1: PTX feed (mg) X2: LPT feed (mg) X3: polymer feed (mg) Response Y1: Y2: Y3: Y4:
PTX ER% LPT ER% LPT DL% PTX DL%
2.50 3.48 150 Expected
Observed
68.30 70.11 1.57 1.09
70.56 70.62 1.58 1.13
Optimization procedure Optimization process was conducted by numerical optimization with Design expertÕ software. PTX EN% was set to be the most important response and LPT EN% was the next optimization periority. PTX in formulation posseses a greater priority since PTX is the main agent and LPT just act as a chemosensetizer. The optimized formulation consisted of 2.50 mg PTX, 3.48 mg LPT and 150.00 mg F127 as primary feeds with 0.782 desirabilty function. PTX EN% and PTX DL% were optimized at 68.30 and 1.09%, while LPT EN% and LPT DL% found to be 70.11 and 1.57%, respectively. The optimized solution was perapared and analyzed for PTX EN%, LPT EN, PTX LD% and LPT LD%. Observed responses were reproducible and they were in agreement with predicted values (Table 4). Particles size measurements and morphology study The size of PTX–LPT-loaded micelles is an important parameter for EPR effect and escape from reticulo-endothelial system30. Mean size of the optimized micelle was found to be 64.81 nm which is slightly larger than PTX loaded P123-F127 micelles (Figure 4a). Dual loading of micelle by PTX and LPT simultaneously might be responsible for this slight size expansion37,47. Polydispersity index (PI) determined to be 0.309. Zeta potential of optimized micelle was around 13.7 mV which might be due to
the partial negative charges of oxygen atoms in pluronic structure (Figure 4b). TEM microscopy analysis (Figure 5) revealed that PTX– LPT-loaded micelles are spherical in shape and possess good morphological characteristics. According to TEM analyses, micelles were spherical in shape with a good core–shell structure. Also no roughness or sharp edges helping the micelles to escape from the reticulo-endothelial system were observed30. It is reported that the spiky nanoparticles could stimulate macrophages cells within the tissue48. The size was about 40 nm (5100 nm) allowing the possibility to be accommodated in endocytic vesicles and access to target cells via endocytosis41. Gray spots represent the PPO core of micelles which encapsulate PTX and LPT. Size of the micelles in TEM microscopic images was about 40 nm which is slightly smaller than results obtained from laser scattering method (64.8 nm). Since in laser scattering method, the hydrodynamic size of the micelles has been measured, size of the micelles is slightly overestimated. In vitro release of PTX and LPT from drug loaded micelles In vitro release profile of PTX–LPT-loaded micelle is shown in Figure 6. In the first 2 h of study both PTX and LPT showed a burst release of 43 and 24%, respectively. After initial burst release, both drugs demonstrated a sustain release pattern during the next 25 h. LPT in comparison to PTX released more slowly
DOI: 10.3109/10837450.2014.965323
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Figure 4. (a) Size distribution and (b) zeta potential.
Figure 5. TEM image.
Figure 6. In vitro release profile of PTX and LPT in aqueous medium at 37 C. Each represented as average ± SD (n ¼ 3).
perhaps due to its more hydrophobic nature (log P was 3.5 and 5.3 for PTX and LPT, respectively)49,50. It seems that more hydrophobic interactions of LPT with core of the micelles caused slower release of LPT. Slower release of LPT in comparison to PTX may not disturbs the LPT efficacy since in this study LPT just acted as a chemosensetizer in the newly developed formulation. After 30 h both PTX and LPT reached the plateau phase. Since further samplings after 30 h of release study showed that neither PTX nor LPT was released to the medium, therefore it was assumed that the plateau phase was reached. After reaching to the plateau phase, it seems that about 20 and 40% of PTX and LPT, respectively, were still entrapped into the micelles. Moreover, the HPLC analysis of micellar system after release experiment confirmed that aforementioned amounts of both PTX and LPT were remained in the system. Since both PTX and LPT are hydrophobe, it seems that hyrdrophobic interactions among drugs and core of the micelle prevent the complete release.
Most probably due to the more hydrophobic interaction among LPT-polymer compared to PTX-polymer, entrapped amount of LPT was higher than PTX in plateau phase. In vitro cytotoxicity assay In vitro cytotoxicity of PTX–LPT-loaded micelles was evaluated compared to the binary mixture of PTX and LPT dissolved in DMSO (Figure 7a). T-47D cell line was selected as metastatic breast cancer in vitro model51. T-47D cells were highly resistant to PTX due to long periods of exposure to PTX, prior to cytotoxicity assay. Development of resistance to PTX is mainly due to the overexpression of P-gp transporters52. Figure 7a shows that PTX–LPT-loaded micelles inhibit the proliferation of T-47D cell line more effectively compared to binary mixture of PTX and LPT. IC50 values were 0.6 ± 0.1 mg/ml for PTX–LPT-loaded micelles and 6.7 ± 1.2 mg/ml for binary mixture of PTX and LPT
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Figure 7. In vitro cytotoxicity assay on T-47D cells (a) PTX–LPT loaded micelles compared to binary mixture of PTX and LPT (b) Pluronic F127.
(Figure 7a). Increasing the concentration of PTX–LPT-loaded micelles clearly reduced the cell viability as suggested by literature53. Also Pluronic F127 exhibited good biocompatibility since cell viability was high in 0.1–1000 mg/ml concentration range of Pluronic F127 (Figure 7b). As seen in this Figure 7(a), PTX–LPT micelles had superior toxic effect in comparison with bare PTX–LPT binary solution with the same concentrations. This superior toxic effect most probably comes from the different cell trafficking mechanism of such a system. Cancerous cells uptake micellar complexes via endocytosis which might be responsible for higher toxicity of formulation compared to binary mixture of PTX and LPT54. Furthermore pluronic F127 can cause ATP depletion in cancerous cell which disturbs the function of P-gp transporters32. Such an inhibition can cause enhanced cytotoxicity of developed formulation. It is reported that LPT sensitizes cancerous cells by the inhibition of efflux pumps which consequently enhance the cytotoxic effect of developed formulation26. Also such an effect was reported for OVCAR-3 and DU145-TXR cell lines in ovarian and prostate cancer, respectively. However, more in vitro studies are required to analyze the impact of LPT-mediated sensitization and consequent PTX enhanced cytotoxicity.
Conclusion The aim of this study was preparation of PTX–LPT-loaded micelles for simultaneous delivery of these two agents. Experimental design and optimization process were carried out by D-optimal method. EN% optimized at 68.30 and 70.11% for PTX and LPT, respectively. Size of the optimized formulation was 64.81 nm with 0.309 PI which confirmed that the micelles possess appropriate morphological characteristics. Release analysis revealed 25 h sustain release pattern after minimal initial burst release. In vitro cytotoxic evaluation of PTX–LPT-loaded micelles was assessed by MTT assay on T-47D cell line. Results suggest that PTX–LPT-loaded micelles possess a higher cytotoxicity effect compared to binary mixture of PTX and LPT. In vitro cytotoxicity studies provoked us for in vivo analysis in our further researches.
Declaration of interest The authors report no declaration of interest.
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36. Mitchell TJ. An algorithm for the construction of ‘‘D-optimal’’ experimental designs. Technometrics 1974;16:203–210. 37. Wei Z, Hao J, Yuan S, et al. Paclitaxel-loaded pluronic P123/F127 mixed polymeric micelles: formulation, optimization and in vitro characterization. Int J Pharm 2009;376:176–185. 38. Chen L, Sha X, Jiang X, et al. Pluronic P105/F127 mixed micelles for the delivery of docetaxel against Taxol-resistant non-small cell lung cancer: optimization and in vitro, in vivo evaluation. Int J Nanomed 2013;8:73–84. 39. D’Souza SS, DeLuca PP. Methods to assess in vitro drug release from injectable polymeric particulate systems. Pharma Res 2006;23: 460–474. 40. Liu Y, Sun J, Cao W, et al. Dual targeting folate-conjugated hyaluronic acid polymeric micelles for paclitaxel delivery. Int J Pharma 2011;421:160–169. 41. Kabanov AV, Batrakova EV, Alakhov VY. PluronicÕ block copolymers as novel polymer therapeutics for drug and gene delivery. J Control Release 2002;82:189–212. 42. Song X, Zhao Y, Hou S, et al. Dual agents loaded PLGA nanoparticles: systematic study of particle size and drug entrapment efficiency. Eur J Pharm Biopharm 2008;69:445–453. 43. Song X, Zhao Y, Wu W, et al. PLGA nanoparticles simultaneously loaded with vincristine sulfate and verapamil hydrochloride: systematic study of particle size and drug entrapment efficiency. Int J Pharm 2008;350:320–329. 44. Wang Y, Yu L, Han L, et al. Difunctional pluronic copolymer micelles for paclitaxel delivery: synergistic effect of folate-mediated targeting and pluronic-mediated overcoming multidrug resistance in tumor cell lines. Int J Pharm 2007;337:63–73. 45. Patil Y, Sadhukha T, Ma L, Panyam J. Nanoparticle-mediated simultaneous and targeted delivery of paclitaxel and tariquidar overcomes tumor drug resistance. J Control Release 2009;136: 21–29. 46. Wong HL, Bendayan R, Rauth AM, Wu XY. Simultaneous delivery of doxorubicin and GG918 (Elacridar) by new polymer-lipid hybrid nanoparticles (PLN) for enhanced treatment of multidrug-resistant breast cancer. J Control Release 2006;116:275–284. 47. Govender T, Stolnik S, Garnett MC, et al. PLGA nanoparticles prepared by nanoprecipitation: drug loading and release studies of a water soluble drug. J Control Release 1999;57:171–185. 48. Albanese A, Sykes EA, Chan WC. Rough around the edges: the inflammatory response of microglial cells to spiky nanoparticles. ACS Nano 2010;4:2490–2493. 49. Yoncheva K, Calleja P, Agu¨eros M, et al. Stabilized micelles as delivery vehicles for paclitaxel. Int J Pharm 2012;436:258–264. 50. Zhang L, Fan C, Guo Z, et al. Discovery of a potent dual EGFR/ HER-2 inhibitor L-2 (selatinib) for the treatment of cancer. Eur J Med Chem 2013;69:833–841. 51. Sartorius CA, Groshong SD, Miller LA, et al. New T47D breast cancer cell lines for the independent study of progesterone B-and A-receptors: only antiprogestin-occupied B-receptors are switched to transcriptional agonists by cAMP. Cancer Res 1994;54: 3868–3877. 52. Gottesman MM, Fojo T, Bates SE. Multidrug resistance in cancer: role of ATP-dependent transporters. Nat Rev Cancer 2002;2:48–58. 53. Zhang W, Shi Y, Chen Y, et al. Multifunctional pluronic P123/F127 mixed polymeric micelles loaded with paclitaxel for the treatment of multidrug resistant tumors. Biomaterials 2011;32:2894–2906. 54. Cho K, Wang X, Nie S, Shin DM. Therapeutic nanoparticles for drug delivery in cancer. Clin Cancer Res 2008;14:1310–1316.
RESEARCH ARTICLE
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Pluronic F127 polymeric micelles for co-delivery of paclitaxel and lapatinib against metastatic breast cancer: preparation, optimization and in vitro evaluation Pooya Dehghan Kelishady, Ebrahim Saadat, Fatemeh Ravar, Hamid Akbari, and Farid Dorkoosh Department of Pharmaceutics, Faculty of Pharmacy, Tehran University of Medical Science, Tehran, Iran
Abstract
Keywords
The aim of this study was to develop and characterize the paclitaxel (PTX)-lapatinib (LPT) loaded micelles for simultaneous delivery against metastatic breast cancer. Efflux pumpmediated drug resistance influences the efficacy of chemotherapeutic regimens. However, in the newly developed delivery system, LPT was selected to act as chemosensetizer. LPT increases the intracellular level of PTX by inhibition of efflux pumps. Pluronic F127 was selected for the preparation of the micelles, and its critical micelle concentration was determined to be 0.012 mg/ml. D-optimal design was used to analyze the impact of different experimental parameters on PTX and LPT encapsulation ratio. PTX encapsulation ratio was optimized at 68.3%, while LPT encapsulation ratio found to be 70.1%. Transmission electron microscope analyses demonstrate that micelles possess a good core–shell structure without any sharp edge. Laser scattering method results indicated that size of the optimized micelles is 64.81 nm with acceptable polydispersity index (0.309). In vitro release studies showed a sustain release pattern. PTX–LPT-loaded micelles suppressed the proliferation of resistant T-47D cell line (IC50 ¼ 0.6 ± 0.1 mg/ml) compared to binary mixture of PTX and LPT (IC50 ¼ 6.7 ± 1.2 mg/ml). Therefore, it is concluded that the developed formulation might increase the therapeutic efficacy in drug resistant metastatic breast cancer.
D-optimal design, drug resistance, efflux pump, lapatinib, paclitaxel, polymeric micelle
Introduction Paclitaxel (PTX) is a common chemotherapeutic agent which is widely applied for the treatment of different types of solid tumors1. PTX suppresses cancer cells proliferation by disturbing microtubule network during the cell division1. PTX also triggers apoptosis (programmed cell death) of cancerous cells by G2/M cell-cycle arrest2,3. However, PTX restricts the proliferation of normal cells which leads to severe side effects decreasing patients compliance during chemotherapy period4. Unfortunately, poor water solubility of PTX (0.3 mg/ml) is a major obstacle upon application of PTX5. In the commercial formulation of PTX, TaxolÕ , the solubility is enhanced by Cremophor ELÕ as a solubilizing agent. However, several studies reported serious side effects of Cremophor ELÕ including hypersensitivity, nephrotoxicity and neurotoxicity6. There are two main novel formulations for PTX, AbrexaneÕ and Genexol-PMÕ , which have been developed as alternatives for TaxolÕ in chemotherapeutic regimens. AbrexaneÕ is an albumin bound PTX nanoparticle which has fewer and limited side effects compared to TaxolÕ 7. However, the high-cost level of AbrexaneÕ restricts its use in clinic8. Genexol-PMÕ (Samyang Co., Seoul, Korea) is the polymeric micelle form of PTX developed by PEG-PLA co-polymer9.
Address for correspondence: Dr. Farid Dorkoosh, Department of Pharmaceutics, Faculty of Pharmacy, Tehran University of Medical Science, Tehran, Iran. E-mail: [email protected]
History Received 29 April 2014 Revised 8 August 2014 Accepted 6 September 2014 Published online 29 September 2014
Several drug delivery systems (DDS) have been designed to overcome PTX solubility problem as well as minimizing PTX side effects on normal cells. DDS including polymeric micelles10,11, liposomes12,13, nanospheres14, nanoparticles15, polymer conjugates16 and dendritic polymers17,18 are all designed for delivery of PTX. In these series of studies, the aqueous solubility of PTX is enhanced to some extent but the resistance of cancerous cells is still a challenging issue. Multidrug resistance (MDR) is one of the main complications which could be developed by cancerous cells. Efflux-mediated drug resistance is mainly provoked by ATP-binding cassette (ABC) family including P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP)19. Efflux pumps decrease the intracellular level of the cytotoxic agents and, as a result, restrict the therapeutic efficacy. A strategy that has been investigated to overcome the efflux-mediated drug resistance is to co-administer a P-gp inhibitor along with the anticancer drug. However, P-gp inhibitors can aggravate the side effects. Therefore, among the alternatives for efflux pump inhibitors, the benefits of chemotherapeutic agents which reduce cancer cells proliferation and inhibit the efflux pumps simultaneously are two-fold. Lapatinib (LPT) is a dual tyrosine kinase inhibitor which targets both human epidermal growth factor receptors (EGFR) and HER2 receptors20. Clinical data demonstrate that LPT monotherapy would be helpful in HER2 positive breast cancer21. In metastatic breast cancer, PTX and LPT combination therapy is one of the main therapeutic regimens clinically
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applied22,23. Recently, some reports demonstrated that tyrosine kinase inhibitors such as LPT act as an efflux pump inhibitor besides of their cytotoxic properties. LPT binds to the internal domain of efflux pump and inhibits the function of transporter therefore sensitizes the MDR cancerous cells to chemotherapeutic agents such as PTX24–26. Since LPT increases intracellular level of PTX by inhibiting efflux pumps, designing a DDS for co-delivery of PTX and LPT would be a good choice (Figure 1). DDS such as polyelectrolyte nanocapsules27 and lipopolymer micelles28 have been developed to deliver PTX and LPT together for treatment of ovarian and prostate cancer, respectively. PTX–LPT dual delivery might be efficient in treatment of metastatic breast cancer since LPT combined with PTX offers a significant and clinically meaningful survival advantage over PTX alone in patients with HER2-positive metastatic breast cancer23. Polymeric micelles have been widely used to enhance the solubility of hydrophobic drugs. Core–shell structure of the micelles enables them to encapsulate the hydrophobic agents, while the hydrophillic shell enhances the stability of the particle. Tumor tissues possess a leaky vascularized network which allows large polymeric complexes to easily overpass the endothelial layer and accumulate in the tumor cells29. This phenomenon is called enhanced permeability and retention (EPR) effect. EPR allows the polymeric micelles to target passively the tumor cells. Protection from inactivation in biological media and long circulation period which enhance the pharmacokinetics of PTX and LPT are the other advantages of polymeric micelles30,31. Pluronic di-block polymers are made of hydrophilic poly ethylene oxide (PEO) and hydrophobic poly propylene oxide (PPO) arranged in di-block structure. Amphiphilic nature of this polymer makes it an appropriate choice for preparation of polymeric micelles. Pluronic polymers induce ATP depletion and consequently cause P-gp inhibition in MDR cells32. Pluronic F127 (PEO100–PPO69–PEO100) is commonly used to improve the stability and encapsulation efficiency of micelles. F127 was selected in order to prepare the micelles due to its biocompatibility and its approval by FDA. The purpose of this study was the development of F127 polymeric micelles encapsulating PTX and LPT as chemotherapeutic agents. Critical micelle concentration (CMC) of Pluronic F127 was determined for assuring that micelles would maintain their form in diluted conditions such as biological media. Encapsulation ratio (EN%) for PTX and LPT was modeled and optimized using response surface methodology approach. Optimized formulation was selected according to PTX and LPT EN%. Morphology of PTX–LPT-loaded micelles was studied by Transmission Electron Microscopy (TEM). Laser scattering method revealed the characteristics of micelles such as shape, form and size. In vitro release test defined the release Figure 1. Aim of design of dual delivery system for PTX and LPT.
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pattern of PTX and LPT from polymeric micelles. Finally, PTX–LPT-loaded micelle’s cytotoxicity was evaluated by MTT assay on resistant T-47D cell line as breast cancer model. In vitro cytotoxicity assay promoted us to expand our studies to in vivo tests in future studies which can determine efficacy of DDS in animal models.
Materials and methods Materials LPT ditosylate (99.9% purity) was purchased from Beijing Mesochem Technology Co. (Beijing, China) and used without further purification. PTX (99% purity) was obtained from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Pluronic F127 was purchased from Gilson Co. (Villiers-le-Bel, France). T-47D cell line was obtained from National Cell Bank of Iran (NCBI), Pastor Institute of Iran. 3-(4,5-Dimethyl-thiazol-2yl)-2,5-diphenyl-tetrazolium bromide (MTT) was purchased from Sigma (St. Louis, MO). Penicillin–streptomycin, RPMI 1640, fetal bovine serum (FBS) and 0.25% (w/v) trypsin–0.03% (w/v) EDTA solution were purchased from Gibco BRL (Gaithersburg, MD). All other solvents and reagents were either analytical or chromatographic grade and used as received. Critical micelle concentration determination In order to determine critical micelle concentration (CMC) of Pluronic F127 in deionized (DI) water, pyrene probe method was applied33. Briefly, pyrene method determines the CMC by analyzing pyrene content incorporating into the micelles. In concentrations around the CMC value, there is a sharp decrease in pyrene spectroflourimetry signals due to its incorporation to the micelles. 5 mg of pyrene was dissolved in acetone and serially diluted to 6.0 106 M using A grade volumetric flasks and A grade volumetric pipettes. 1 ml aliquot of the pyrene solution was added into 10 ml volumetric flasks. Acetone content of each flask was evaporated completely under mild heating. Each flask was filled up with 10 ml of F127 dispersions in DI water (concentrations ranging over 5 104 to 5 mg/ml). The final concentration of pyrene was controlled at 6.0 107 M. The solutions were sonicated for 30 min and then stirred at 700 rpm overnight in the room temperature. The spectroflourimetry analyses were performed by a spectroflourimeter (Shimizu, Tokyo, Japan). Pyrene was excited at 334 nm, and its emission was recorded at 373 and 384 nm, which corresponds to the first and third vibrational peaks, respectively. Excitation and emission slits set at 8 and 2 nm, respectively. The intensity ratio of the first peak (I1, 373 nm) to the third peak (I3, 384 nm) was plotted against the polymer concentration and analyzed for the calculation of CMC.
PTX–LPT-loaded pluronic polymeric micelles
DOI: 10.3109/10837450.2014.965323
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Preparation of PTX–LPT-loaded micelles PTX–LPT-loaded micelles were prepared by thin-film hydration method34. Various defined amounts of PTX, LPT and F127 were dissolved in 10 ml acetonitrile using a round bottom flask. Acetonitrile was evaporated by rotary evaporator at about 50 C. Residual acetonitrile content was evaporated under the vacuum condition overnight in order to reassure that organic solvent was eliminated from final formulation completely. In the following day, thin film was hydrated by 10 ml of distilled water at 60 C and stirred for 30 min to prepare the micellar solution. The solution was filtrated through 0.22 mm cellulose acetate (CA) filter to eliminate unincorporated drug aggregates from the final micellar solution. Obtained micellar solution was lyophilized and stored in 5 C for further drug content analysis. Drug content analyses and HPLC procedure PTX and LPT contents were analyzed by HPLC system (Agilent Technologies model 1260 Infinity) which was equipped with an autosampler system (Agilent Technologies, Santa Clara, CA) fitted with a 25-ml sample loop and G1315D diode array detector (Agilent Technologies, Santa Clara, CA). A reversedphase column (C18 MZ-Analytical column, 5 mm, 150 4.6 mm, OSD-3) which was protected by pre-column (5 mm, 4.0 4.6 mm, OSD-3) was used at room temperature. Chromatographic data was analyzed by Agilent Chemostation for LC system software (B.04.02). The mobile phase consisted of acetonitrile and deionized water (70/30, V/V). Mobile phase was freshly prepared for each run and filtered through PTFE filter (0.45 mm). Injection volume was set at 20 ml, and flow rate was adjusted at 1.5 ml/min. Detection wave length was monitored at 227 nm. The lyophilized PTX–LPT micelles were hydrated by DI water. 1 ml of micellar solution was mixed with the equal volume of acetonitrile and sonicated in bath sonicator for 10 min in order to disturb the micelle structure. 20 ml of extracted solution was injected into the HPLC to analyze PTX and LPT incorporated into the micelles. Typical retention times for PTX and LPT were found out to be 2 and 4 min, respectively. The concentrations of PTX and LPT were determined by comparing the peak areas with the standard calibration curve.
Experimental design D-optimal design D-optimal method was conducted to figure out the effect of major parameters on the micelle’s characteristics in a few experimental runs35,36. Major micelle’s characteristics which are defined as responses are mainly depended on different experimental parameters in various degrees of interaction. D-optimal design was used to analyze the impact of three different major parameters, including feeding PTX (X1), feeding LPT (X2) and feeding F127 (X3). Both PTX and LPT feeds were ranged over 1 to 5 mg, while the polymer feed was selected to be 50–150 mg. Former studies on similar formulations and also results of some preliminary experiments were used in order to select the major parameters and their levels in the experimental design37,38. PTX encapsulation ratio (PTX EN%, Y1) and LPT encapsulation ratio (LPT EN%, Y2) were calculated, and their surface response was analyzed because they were generally regarded as the major characteristics of developed polymeric micelles. Encapsulation ratios and also drug-loading coefficients (DL%) were calculated using the following equations:
ER% ¼
DL% ¼
3
weight of the drug in micelles 100 weight of the feeding drug
weight of the drug in micelles 100 weight of the feeding polymer and drug
Experimental designs, analysis and optimization were performed using Design expertÕ (version 7.0.0, stat-Ease, Inc., Minneapolis, MN). Eighteen experimental runs were conducted using D-optimal design and two responses were obtained for further assessments. D-optimal design, as a technique, assists the selection of the model which can precisely and accurately define parameter–response relationship. According to the previous studies, quadratic and 2FI models mainly define this relationship and are introduced as follows: Yn ¼ b0 þb1 X1 þb2 X2 þb3 X3 þ . . . þb12 X1 X2 þb13 X X3 þ . . . þb11 X12 þb22 X22 þb33 X32 ðQuadratic modelÞ Yn ¼ b0 þb1 X1 þb2 X2 þb3 X3 þ . . . þb12 X1 X2 þb13 X X3 ð2FI modelÞ In both 2FI and quadratic models, X1 to X3 represent individual effect of parameter, whereas X1X2 and other combination of factors show the binary interactions of parameters on each other. X 21 to X 23 demonstrate second-order interaction of parameters on the response surface in the quadratic model. Significancy of the models and their coefficients were investigated by Analysis of Variance (ANOVA). Optimization procedure Optimization procedure was performed using numerical optimization by Design expertÕ (version 7.0.0, stat-Ease, Inc., Minneapolis, MN). In brief, our goal was to maximize the encapsulation ratio for PTX and LPT. In practice, it is unlikely to maximize all parameters simultaneously. Therefore, numerical optimization method was performed which is based upon utilization of desirability functions. Each single response has its own desirability (d1 to d2). Individual desirability can be used to calculate the overall desirability by below equation in which D represents overall desirability and d1 to d2 is corresponding to individual desirabilities: D ¼ðd1 d2 Þ1=2 Overall desirability function represents the overall optimization state. Design expertÕ nominates the best solutions; we selected the best formulation according to the overall desirability function and also with respect to individual desirabilities. Particles size measurements and morphology study Size, polydispersity index (PI) and zeta potential measurements were conducted by Malvern zetasizer (Malvern Instruments Ltd, Malvern, UK). The analyses were performed with 5mWHe–Ne laser (632.8 nm) at a scattering angle of 90 at 25 C. Lyophilized powder was hydrated with 10 ml DI water. For appropriate size analysis and avoiding multi-scattering phenomena, obtained micellar solution was diluted in 10 ml of DI water. Afterwards, samples were vortexed well and placed in quartz cuvette for size measurements. Morphology studies of PTX–LPT-loaded micelles
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were carried out by Transmission Electron Microscope (TEM). Prior to visualization, samples were diluted and fixed on a grid for microscopic analyses.
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In vitro release of PTX and LPT from drug-loaded micelles In vitro release of the optimized PTX–LPT-loaded micelles was performed in an aqueous solution using dialysis method in sink condition39. 1 ml PTX–LPT-loaded micelle solution was added to a dialysis bag [molecular weight cutoff (MWCO) ¼ 12 000 Da]. Dialysis bag was end-sealed completely and fully immersed in 100 ml PBS (pH 7.4) containing PEG 400, 10% (V/V). PEG 400 added to enhance the solubility of PTX and maintain the sink condition5,40. Release medium was maintained at 37 ± 0.5 C and stirred at 100 rpm for 48 h. At predetermined time intervals, 1 ml aliquot was withdrawn for further analyses. Instantly after sampling, entire release medium was substituted by fresh medium for avoiding PTX and LPT accumulation in medium and maintaining the sink condition. Samples were analyzed by RP-HPLC method discussed earlier, and the amount of released PTX and LPT was determined. Finally, the cumulative release of PTX and LPT was plotted against time to evaluate the in vitro release profiles. In vitro cytotoxicity assay Resistant T-747D cell line which is derived from mammary glands’ metastatic sites was selected as a model for metastatic breast cancer. T-47D was cultured at 37 C and 5% CO2. Culture medium consisted of RPMI 1640 medium supplemented by 10% FBS, 100 IU/ml penicillin and 100 mg/ml streptomycin sulfate. Cells were seeded at the density of 5 103 cells per well in 96-well plates. After 24 h of incubation at 37 C with 5% CO2, the growth medium was replaced by 200 ml medium containing Pluronic F127, binary mixture of PTX and LPT in DMSO and PTX–LPT-loaded micelles in concentrations ranging from 0.001 to 1000 mg/ml. After 72 h incubation, cell survival was measured using tetrazolium salt (MTT) assay. 180 ml of fresh growth medium and 20 ml of MTT (5 mg/ml) solution were added to each well. The plate was incubated for an additional 4 h, and then 200 ml of DMSO was added to each well to dissolve any purple formazan crystals formed. The plates were vigorously shaken before measuring the relative color intensity. The absorbance at 570 nm of each well was measured by a microplate reader (BioTek ELx 800, USA). Each assay was repeated in triplicates.
Results and discussion CMC determination Critical micelle concentration is an important factor which reveals the stability of micelle both in vivo and in vitro. Micelles may disassemble in diluted media and it is an important issue especially in DDS. When the micelles are administered intravenously, they are being diluted several times in blood stream. If the CMC value of micelle is low enough, it means that micelle structure remains intact even after several dilutions. On the other hand, micelles with high-CMC values may be disassembled and immaturely release their payload which can cause toxicity and other side effects. Pyrene method is a simple procedure for CMC determination. Pyrene incorporates into the micelles due to its hydrophobe nature, while the unincorporated pyrene remains in the aqueous medium and demonstrate florescence properties of pyrene. Ratio of the first vibrational peak (I1, 373 nm) to the third vibrational peak (I3, 384 nm) indicates the unincorporated pyrene content. Around CMC value because of initiation of micelle forming process, pyrene starts to incorporate into the micelles and consequently a rapid I1/I3 decrease is observed.
Figure 2. Plot of I1/I3 versus concentration of F127 in DI water.
Plotting I1/I3 against logarithm of polymer concentration shows two different bunches of points in the plot: high I1/I3 group and low I1/I3 group (Figure 2). Two regression lines were plotted by these two groups of points. The interaction point of these two lines indicates the CMC value. CMC value found to be 0.012 mg/ml which is relevant with previously measured CMC value by iodine UV spectrometry method37. Measured CMC value indicates that F127 polymer possesses a good micelle forming capability compared to the other pluronic polymers which make it suitable for preparation of the polymeric micelles37,41. Design of experiment D-optimal design Eighteen experimental runs (Table 1) were conducted by D-optimal design to analyze and model the impact of feeding PTX (X1), feeding LPT (X2) and feeding F1127 (X3) on the PTX encapsulation ratio (PTX EN%, Y1) and LPT encapsulation ratio (LPT EN%, Y2). PTX encapsulation ratio has ranged from 2.82 to 88.90%. This wide response range demonstrates that PTX encapsulation ratio is strongly depended on experimental parameters. Drug loading coefficient for PTX also has ranged from 0.22 to 1.43%. Statistical analysis shows that PTX encapsulation ratio fitted into a quadratic model [Equation (1)] with significant p value (p value50.05) and also insignificant lack of fit (p value40.05) (Table 2). PTX EN% ¼ 59:83114 43:00824 PTX þ 44:30701 LPT 0:29956 polymer þ 5:0918 PTX2 7:23246 LPT2 þ 2:53353 3
10 polymer2 þ 0:29835 PTX LPT
ð1Þ
6:17912 103 PTX polymer þ 9:99386 103 LPT polymer F127 Polymer feed has increased EN% for both PTX and LPT since it increases the amount of polymer available for encapsulating the hydrophobe PTX and LPT. A significant (p value50.05) first-order impact of polymer is suggested for PTX EN% and LPT EN%. According to the literature, similar result was reported for encapsulation of vincristine into PLGA nanoparticles42,43. First- and second-order impact of PTX feed on PTX EN% was
PTX–LPT-loaded pluronic polymeric micelles
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Table 1. D-optimal experimental runs.
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Experimental parameters (mg) Std
Run
PTX feed
LPT feed
Polymer feed
PTX encapsulation
LPT encapsulation
LPT loading
PTX loading
11 12 16 2 15 13 8 7 5 9 4 18 10 14 3 17 6 1
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
4.00 1.00 1.00 1.00 1.00 5.00 1.00 3.00 1.00 5.00 5.00 1.00 3.00 5.00 5.00 5.00 3.00 1.00
3.00 5.00 1.00 1.00 5.00 5.00 3.00 3.00 1.00 1.00 1.00 1.00 5.00 5.00 5.00 1.00 1.00 5.00
75.00 50.00 50.00 150.00 150.00 50.00 100.00 150.00 50.00 50.00 150.00 150.00 50.00 150.00 100.00 150.00 100.00 150.00
21.14 62.52 53.72 70.45 88.9 16.8 85.86 75.19 50.01 2.52 24.6 69.11 6.47 27.45 26.06 12.44 11.95 75.11
83.95 16.68 45.81 53.14 72.75 48.24 51.81 60.54 46.14 63.89 75 51.95 11.33 78.72 69.42 52.55 77.53 75.68
3.07 1.49 0.88 0.35 2.33 4.03 1.49 1.16 0.88 1.14 0.48 0.34 0.97 2.46 3.4 0.33 0.73 2.42
1.03 1.12 1.03 0.46 0.56 1.4 0.82 1.43 0.96 0.22 0.79 0.45 0.33 0.86 1.18 0.4 0.34 0.48
Table 2. D-optimal analysis of PTX EN%.
Source Model X1 X2 X3 X21 X22 X23 X1X2 X1X3 X2X3 Residual Lack of fit Pure error Cor total
Response (%)
Table 3. D-optimal analysis of LPT EN%.
Sum of SQUARES
Degree of freedom (df)
Mean squares
14100.53 7605.20 447.68 1531.02 873.23 1427.73 84.45 17.31 4.26 12.14 791.34 614.54 176.79 14891.87
9 1 1 1 1 1 1 1 1 1 8 4 4 17
1566.73 7605.20 447.68 1531.02 873.23 1427.73 84.45 17.31 4.26 12.14 98.92 153.64 44.20
F value
p value*
Source
Sum of squares
Degree of freedom (df)
Mean squares
15.84 76.88 4.53 15.48 8.83 14.43 0.85 0.18 0.043 0.12
0.0003 50.0001 0.0661 0.0043 0.0178 0.0052 0.3825 0.6867 0.8407 0.7351
Model X1 X2 X3 X1X2 X1X3 X2X3 Residual Lack of fit Pure error Cor total
4983.84 1385.41 203.71 1738.12 5.12 301.83 2074.64 1884.82 1627.76 257.06 6868.66
6 1 1 1 1 1 1 11 7 4 17
830.64 1385.41 203.71 1738.12 5.12 301.83 2074.64 171.35 232.54 64.26
3.48
0.1274
*Significant at 0.05 level.
significant (p value50.5). Higher PTX EN% is achieved for lower PTX feeding (Figure 3). There is a limited capacity in the core part of pluronic micelle which would be saturated by defined amount of PTX. Therefore, by increasing the total amount of PTX feed, the micelle core would reach to the saturation which leads to decrease in EN% of PTX accordingly. As it is reported, hydrophobic interactions among PTX molecules are greater than PTX and Pluronics44. Therefore, due to the strong hydrophobic interaction among PTX molecules and low solubility of PTX in water, unincorporated PTX aggregates would eventually form in the medium. These drug aggregates would limit available free PTX for encapsulating into the micelles and consequently reduce PTX EN%. PTX EN% is being maximized in medium concentrations of LPT ranging from 2 to 4 mg. The model suggests a significant second-order relation (p-value50.05). It is clear that large amounts of LPT would form unincorporated LPT aggregates that may adsorb free PTX in the medium. In analogous study focusing on PTX and tariquidar co-delivery systems, an increase in amount of tariquidar has resulted a reduction in PTX EN%45.
F value
p value*
4.85 8.09 1.19 10.14 0.030 1.76 12.11
0.0117 0.0160 0.2989 0.0087 0.8659 0.2113 0.0052
3.62
0.1157
*Significant at 0.05 level.
On the other hand, LPT EN% has decreased in small LPT feeds. The formation of drug aggregates in small feeding amounts of LPT is negligible; consequently, LPT freely entrapped and fill the core of micelles before PTX due to its more hydrophobe nature. Such a decrease of EN% is also reported in doxorubicinelacridar nanoparticles46. In conclusion, LPT feeds higher than 4 mg and lower than 2 mg would limit PTX EN%. LPT EN% has ranged from 11.83 to 83.95% which is apparently a wide range. Drug loading coefficient for LPT has also ranged from 0.33 to 4.03%. LPT EN% is analyzed by a significant 2FI model (p-value50.05) with insignificant lack of fit (p-value40.05) (Table 3) as follows: LPT EN% ¼ 49:87694 þ 9:80502 PTX 15:25792 LPT 2:51603 103 polymer þ 0:16007 PTX LPT 0:051864 PTX polymer þ 0:12885 LPT
ð2Þ
polymer LPT EN% is inversely related to LPT feeding amount (Figure 3) due to the limited encapsulation efficency of polymer and also acceleration of drug aggregates formation in
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Figure 3. Reponse–parameter relationship (a) PTX EN% (b) LPT EN%.
concentrated intial feeds. The increase of the entrapped amount of LPT was not in proportion to the increase of the initial drug content, thus the entrapment efficiency was decreased42. Mean encapsulation ratio for LPT (57.51%) is slightly greater than PTX (43.35%) and it is less depended on the primary feed, and the model shows only a significant first order relation (p-value50.05). Because of more hyrophobic nature of LPT compared to PTX, it seems that LPT has more conviniently entered into the micelle core. Interestingly, PTX feed raises LPT EN%. As mentioned before, PTX EN% is limited by increasing PTX feed. It seems reasonable to state that by decreasing PTX EN%, significant portion of micelle core is being occupied by LPT.
Table 4. The observed and predicted response values for the optimized formulation. Parameter
Optimized level
X1: PTX feed (mg) X2: LPT feed (mg) X3: polymer feed (mg) Response Y1: Y2: Y3: Y4:
PTX ER% LPT ER% LPT DL% PTX DL%
2.50 3.48 150 Expected
Observed
68.30 70.11 1.57 1.09
70.56 70.62 1.58 1.13
Optimization procedure Optimization process was conducted by numerical optimization with Design expertÕ software. PTX EN% was set to be the most important response and LPT EN% was the next optimization periority. PTX in formulation posseses a greater priority since PTX is the main agent and LPT just act as a chemosensetizer. The optimized formulation consisted of 2.50 mg PTX, 3.48 mg LPT and 150.00 mg F127 as primary feeds with 0.782 desirabilty function. PTX EN% and PTX DL% were optimized at 68.30 and 1.09%, while LPT EN% and LPT DL% found to be 70.11 and 1.57%, respectively. The optimized solution was perapared and analyzed for PTX EN%, LPT EN, PTX LD% and LPT LD%. Observed responses were reproducible and they were in agreement with predicted values (Table 4). Particles size measurements and morphology study The size of PTX–LPT-loaded micelles is an important parameter for EPR effect and escape from reticulo-endothelial system30. Mean size of the optimized micelle was found to be 64.81 nm which is slightly larger than PTX loaded P123-F127 micelles (Figure 4a). Dual loading of micelle by PTX and LPT simultaneously might be responsible for this slight size expansion37,47. Polydispersity index (PI) determined to be 0.309. Zeta potential of optimized micelle was around 13.7 mV which might be due to
the partial negative charges of oxygen atoms in pluronic structure (Figure 4b). TEM microscopy analysis (Figure 5) revealed that PTX– LPT-loaded micelles are spherical in shape and possess good morphological characteristics. According to TEM analyses, micelles were spherical in shape with a good core–shell structure. Also no roughness or sharp edges helping the micelles to escape from the reticulo-endothelial system were observed30. It is reported that the spiky nanoparticles could stimulate macrophages cells within the tissue48. The size was about 40 nm (5100 nm) allowing the possibility to be accommodated in endocytic vesicles and access to target cells via endocytosis41. Gray spots represent the PPO core of micelles which encapsulate PTX and LPT. Size of the micelles in TEM microscopic images was about 40 nm which is slightly smaller than results obtained from laser scattering method (64.8 nm). Since in laser scattering method, the hydrodynamic size of the micelles has been measured, size of the micelles is slightly overestimated. In vitro release of PTX and LPT from drug loaded micelles In vitro release profile of PTX–LPT-loaded micelle is shown in Figure 6. In the first 2 h of study both PTX and LPT showed a burst release of 43 and 24%, respectively. After initial burst release, both drugs demonstrated a sustain release pattern during the next 25 h. LPT in comparison to PTX released more slowly
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Figure 4. (a) Size distribution and (b) zeta potential.
Figure 5. TEM image.
Figure 6. In vitro release profile of PTX and LPT in aqueous medium at 37 C. Each represented as average ± SD (n ¼ 3).
perhaps due to its more hydrophobic nature (log P was 3.5 and 5.3 for PTX and LPT, respectively)49,50. It seems that more hydrophobic interactions of LPT with core of the micelles caused slower release of LPT. Slower release of LPT in comparison to PTX may not disturbs the LPT efficacy since in this study LPT just acted as a chemosensetizer in the newly developed formulation. After 30 h both PTX and LPT reached the plateau phase. Since further samplings after 30 h of release study showed that neither PTX nor LPT was released to the medium, therefore it was assumed that the plateau phase was reached. After reaching to the plateau phase, it seems that about 20 and 40% of PTX and LPT, respectively, were still entrapped into the micelles. Moreover, the HPLC analysis of micellar system after release experiment confirmed that aforementioned amounts of both PTX and LPT were remained in the system. Since both PTX and LPT are hydrophobe, it seems that hyrdrophobic interactions among drugs and core of the micelle prevent the complete release.
Most probably due to the more hydrophobic interaction among LPT-polymer compared to PTX-polymer, entrapped amount of LPT was higher than PTX in plateau phase. In vitro cytotoxicity assay In vitro cytotoxicity of PTX–LPT-loaded micelles was evaluated compared to the binary mixture of PTX and LPT dissolved in DMSO (Figure 7a). T-47D cell line was selected as metastatic breast cancer in vitro model51. T-47D cells were highly resistant to PTX due to long periods of exposure to PTX, prior to cytotoxicity assay. Development of resistance to PTX is mainly due to the overexpression of P-gp transporters52. Figure 7a shows that PTX–LPT-loaded micelles inhibit the proliferation of T-47D cell line more effectively compared to binary mixture of PTX and LPT. IC50 values were 0.6 ± 0.1 mg/ml for PTX–LPT-loaded micelles and 6.7 ± 1.2 mg/ml for binary mixture of PTX and LPT
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Figure 7. In vitro cytotoxicity assay on T-47D cells (a) PTX–LPT loaded micelles compared to binary mixture of PTX and LPT (b) Pluronic F127.
(Figure 7a). Increasing the concentration of PTX–LPT-loaded micelles clearly reduced the cell viability as suggested by literature53. Also Pluronic F127 exhibited good biocompatibility since cell viability was high in 0.1–1000 mg/ml concentration range of Pluronic F127 (Figure 7b). As seen in this Figure 7(a), PTX–LPT micelles had superior toxic effect in comparison with bare PTX–LPT binary solution with the same concentrations. This superior toxic effect most probably comes from the different cell trafficking mechanism of such a system. Cancerous cells uptake micellar complexes via endocytosis which might be responsible for higher toxicity of formulation compared to binary mixture of PTX and LPT54. Furthermore pluronic F127 can cause ATP depletion in cancerous cell which disturbs the function of P-gp transporters32. Such an inhibition can cause enhanced cytotoxicity of developed formulation. It is reported that LPT sensitizes cancerous cells by the inhibition of efflux pumps which consequently enhance the cytotoxic effect of developed formulation26. Also such an effect was reported for OVCAR-3 and DU145-TXR cell lines in ovarian and prostate cancer, respectively. However, more in vitro studies are required to analyze the impact of LPT-mediated sensitization and consequent PTX enhanced cytotoxicity.
Conclusion The aim of this study was preparation of PTX–LPT-loaded micelles for simultaneous delivery of these two agents. Experimental design and optimization process were carried out by D-optimal method. EN% optimized at 68.30 and 70.11% for PTX and LPT, respectively. Size of the optimized formulation was 64.81 nm with 0.309 PI which confirmed that the micelles possess appropriate morphological characteristics. Release analysis revealed 25 h sustain release pattern after minimal initial burst release. In vitro cytotoxic evaluation of PTX–LPT-loaded micelles was assessed by MTT assay on T-47D cell line. Results suggest that PTX–LPT-loaded micelles possess a higher cytotoxicity effect compared to binary mixture of PTX and LPT. In vitro cytotoxicity studies provoked us for in vivo analysis in our further researches.
Declaration of interest The authors report no declaration of interest.
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