Rapamycin-loaded polysorbate 80-coated PLGA nanoparticles: Optimization of formulation variables and in vitro anti-glioma assessment

https://doi.org/10.1016/j.jddst.2019.05.026Get rights and content

Abstract

Rapamycin is a promising anticancer agent, but because it has poor water solubility and low bioavailability its clinical use is limited. The aim of this study was to develop rapamycin-loaded poly(lactide-co-glycolide) nanoparticles coated or not with polysorbate 80 for drug delivery to glioma cells. Nanoparticles were prepared using the emulsification–diffusion method, formulated and optimized through Box-Behnken design, and characterized by DLS, SEM, and DSC. In vitro drug release and stability studies were also performed. The anti-glioma activity of the optimized nanoformulations was tested on C6 glioma cells by cytotoxicity assay and cytoskeletal integrity assessment. The respective values for the particle size, polydispersity index, zeta potential, and drug entrapment efficiency of the optimized uncoated and polysorbate 80-coated nanoparticles were 232 vs. 247 nm, 0.059 vs. 0.103, −16.69 vs. −11.76 mV, 43.7 vs. 28.51%. SEM and DSC analyses revealed that rapamycin was entrapped into spherical nanoparticles. These nanoparticles showed a biphasic drug release pattern and exhibited satisfactory storage stability for at least three months. Anti-glioma activity studies demonstrated that the polysorbate 80-coated nanoparticles were more effective than the uncoated ones and the free rapamycin. These findings suggest that these nanoformulations could be useful in glioma chemotherapy.

Introduction

Rapamycin (Rapa), a hydrophobic macrolide derived from the bacterium Streptomyces hygroscopicus, is an immunosuppressive agent clinically approved for preventing organ transplant rejection [1,2]. But recently Rapa was shown to have anticancer properties in several human cancers, including malignant brain gliomas [3,4]. Rapa's anticancer activity consists of the selective inhibition of the mammalian target of rapamycin (mTOR), which regulates cell growth, proliferation, motility, metabolism, and survival [5]. mTOR is found to be over-activated in certain cancer cells, so inhibiting it could promote both apoptotic and autophagic cell death [6]. Therefore, mTOR inhibition is an effective approach for targeted cancer therapy.

Though Rapa has a high potential for treating cancer, several challenges to its clinical applicability must be addressed. These include water insolubility (2.6 μg/mL), low tumor specificity, dose-dependent toxicity, low bioavailability, and rapid systemic clearance [4,7]. Also, Rapa is pumped out of tumor cells by drug efflux transporters (mainly P-glycoprotein) that impede its cellular accumulation [8]. These drug efflux transporters are over-expressed not only in brain glioma cells, but also in endothelial cells of the blood–brain barrier (BBB), which control the entry of numerous drugs into the brain [9,10]. Given these conditions, developing nanoparticulate drug delivery systems could be an excellent strategy for overcoming these problems.

Biodegradable polymeric nanoparticles (NPs) are colloidal particles that range from 1 to 1,000 nm in diameter and are frequently used as drug delivery systems. They can pass through biological barriers, transport drugs to target sites, increase drug solubility and stability, and reduce side-effects [11]. Because of its broad range of properties such as biodegradability and biocompatibility, poly(d,l-lactide-co-glycolide) (PLGA) –an FDA-approved polymer for human applications– has been widely employed in developing NPs that carry hydrophilic or lipophilic anticancer agents [12]. Brain drug delivery based on the use of PLGA NPs has been recently studied for treating malignant gliomas. However, the success or failure of PLGA NPs as brain-targeted drug delivery vehicles are strongly related to the adsorption of proteins from the bloodstream and subsequent formation of the so-called protein corona, whose composition varies depending on the physicochemical properties of NPs [13]. In the case of PLGA NPs, the binding of opsonins such as immunoglobulin G, fibrinogen, and complement factor to the NP surface causes NP sequestration and elimination by the reticuloendothelial system (RES), reducing thus their blood circulation time and preventing their accumulation at the target site [13,14]. To improve the NP in vivo performance, PLGA NPs can be coated with PEG-based non-ionic surfactants like polysorbate 80 (P80). After intravenous administration, P80-coated NPs have been shown to have a protein corona enriched with albumin and apolipoproteins, which can prolong the NP circulation time [15,16]. P80 coating also decreases the interaction between NPs and opsonins and avoids the RES recognition “stealth effect” [16]. Furthermore, the apolipoproteins (especially B and E) have been involved in the success of the P80-coated NPs to cross the BBB and tumors. The P80-coated NPs seem to mimic natural lipoprotein particles due to the apolipoproteins adsorption, and then they interact with the low-density lipoprotein receptors located in the brain capillary endothelial cells and upregulated in the malignant tumor cells [14]. These interactions promote drug delivery into both brain and tumor via receptor-mediated endocytosis [15,17]. In addition, P80 coating could increase drug transport by inhibiting the P-glycoprotein drug efflux system [15]. Kreuter and coworkers have evidenced widely the effectiveness of the P80 coating on brain drug delivery by showing the higher antitumor effect of doxorubicin-loaded P80-coated NPs in glioblastoma models [[17], [18], [19]].

Over the past twenty years, our research group has developed and diversified the uses of the emulsification–diffusion method for preparing NPs from preformed polymers [20,21]. However, formulation parameters need to be optimized to enhance the performance of NPs as targeted drug delivery systems [22]. In this scenario, the statistical design of experiments coupled with the surface response methodology is a powerful and efficient statistical tool for understanding the effect of independent variables on dependent variables in drug formulation development. Using mathematical and graphical models, it also allows the determination of optimal levels of the independent variables required for a desirable response [23].

In this study, we prepared Rapa-loaded PLGA nanoparticles (Rapa-PLGA-NPs) coated with P80 (Rapa-PLGA-P80-NPs) using the emulsification–diffusion method. Furthermore, a Box-Behnken design with response surface methodology was used to optimize the formulation based on their physicochemical characteristics. The optimized NPs were characterized for various physicochemical, morphological, and solid-state properties. The in vitro drug release behavior, storage stability, and in vitro anti-glioma activity of the optimized NPs were determined as well.

Section snippets

Materials

Rapamycin (Rapa: Sirolimus 98%) was generously donated by Grupo Medifarma, S.A. de C.V. (Morelos, Mexico). Poly(d,l-lactide-co-glycolide) acid (PLGA 50:50, DLG 4A, molecular weight ∼38,000) was obtained from Lakeshore Biomaterials (Birmingham, AL, USA). Poly(vinyl alcohol) (PVA: Mowiol® 4–88, molecular weight ∼31,000), polysorbate 80 (P80: Tween®80), dimethylthiazol diphenyltetrazolium bromide (MTT), and phalloidin–tetramethylrhodamine B isothiocyanate (TRITC-phalloidin) were all purchased from

Preparation and optimization of the Rapa-loaded NPs

The emulsification–diffusion method was fruitfully utilized to prepare the Rapa-PLGA-NPs and Rapa-PLGA-P80-NPs in a simple and reproducible way.

Conclusion

In the present study, Rapa-PLGA-NPs and Rapa-PLGA-P80-NPs were successfully prepared by the emulsification–diffusion method and formulated using a three-factor, three-level Box-Behnken design, which suggested that the desirable physicochemical properties of these NPs can be obtained by controlling the formulation variables. In fact, the procedure followed effectively produced NPs with small particle size, narrow size distribution, quasi-neutral charge surface, satisfactory DEE, and good storage

Conflict of interest

The authors declare no conflict of interest.

Acknowledgements

The authors are grateful to Alicia del Real-López (Departamento de Nanotecnología, Centro de Física Aplicada y Tecnología Avanzada; CFATA, Universidad Nacional Autónoma de México) for her collaboration in the scanning electron microscope studies. Oscar Escalona-Rayo acknowledges the support provided by the CONACYT-Mexico [CVU/Becario: 619711/576545]. This work was supported by the PAPIIT/UNAM [2019143]; the CONACyT CB [221629]; the CONACyT INFRA [251940]; and the PIAPI [001].

References (52)

  • M.L. Zambrano-Zaragoza et al.

    Optimization of nanocapsules preparation by the emulsion–diffusion method for food applications

    LWT - Food Sci. Technol. (Lebensmittel-Wissenschaft -Technol.)

    (2011)
  • C.E. Mora-Huertas et al.

    Influence of process and formulation parameters on the formation of submicron particles by solvent displacement and emulsification–diffusion methods critical comparison

    Adv. Colloid Interface Sci.

    (2011)
  • D. Quintanar-Guerrero et al.

    Influence of stabilizing agents and preparative variables on the formation of poly(D,L-lactic acid) nanoparticles by an emulsification–diffusion technique

    Int. J. Pharm.

    (1996)
  • M.J. Galindo-Pérez et al.

    Optimization of the emulsification–diffusion method using ultrasound to prepare nanocapsules of different food-core oils

    LWT - Food Sci. Technol. (Lebensmittel-Wissenschaft -Technol.)

    (2018)
  • R. Rani et al.

    Optimization and evaluation of bioactive drug-loaded polymeric nanoparticles for drug delivery

    Int. J. Biol. Macromol.

    (2015)
  • M.S. Baig et al.

    Application of Box-Behnken design for preparation of levofloxacin-loaded stearic acid solid lipid nanoparticles for ocular delivery: optimization, in vitro release, ocular tolerance, and antibacterial activity

    Int. J. Biol. Macromol.

    (2016)
  • B. Kumar et al.

    Recent advances in nanoparticle-mediated drug delivery

    J. Drug Deliv. Sci. Technol.

    (2017)
  • W. Abdelwahed et al.

    A pilot study of freeze drying of poly(epsilon-caprolactone) nanocapsules stabilized by poly(vinyl alcohol): formulation and process optimization

    Int. J. Pharm.

    (2006)
  • J. Vandervoort et al.

    Biocompatible stabilizers in the preparation of PLGA nanoparticles: a factorial design study

    Int. J. Pharm.

    (2002)
  • S.K. Sahoo et al.

    Residual polyvinyl alcohol associated with poly (D,L-lactide-co-glycolide) nanoparticles affects their physical properties and cellular uptake

    J. Control. Release

    (2002)
  • P. Simamora et al.

    Solubilization of rapamycin

    Int. J. Pharm.

    (2001)
  • H. Keles et al.

    Investigation of factors influencing the hydrolytic degradation of single PLGA microparticles

    Polym. Degrad. Stabil.

    (2015)
  • S. Fredenberg et al.

    The mechanisms of drug release in poly(lactic-co-glycolic acid)-based drug delivery systems–a review

    Int. J. Pharm.

    (2011)
  • G.D. Rosa et al.

    Influence of the co-encapsulation of different non-ionic surfactants on the properties of PLGA insulin-loaded microspheres

    J. Control. Release

    (2000)
  • N. Sharma et al.

    Effect of process and formulation variables on the preparation of parenteral paclitaxel-loaded biodegradable polymeric nanoparticles: a co-surfactant study

    Asian J. Pharm. Sci.

    (2016)
  • J.J. Augustine et al.

    Use of sirolimus in solid organ transplantation

    Drugs

    (2007)
  • Cited by (35)

    • Nose to Brain Delivery of Transferrin conjugated PLGA nanoparticles for clonidine

      2023, International Journal of Biological Macromolecules
    • Optimization, characterization and in vivo evaluation of mupirocin nanocrystals for topical administration

      2022, European Journal of Pharmaceutical Sciences
      Citation Excerpt :

      The desirability function technique was then used to optimize the formulation using numerical and graphical analysis. A desirability value close to 1 corresponds to the desired response (Oscar et al., 2019). Following selection, the optimal MP-NC were subjected to further investigations.

    • Potential targeting sites in brain and brain tumors

      2022, Nanocarriers for Drug-Targeting Brain Tumors
    View all citing articles on Scopus
    View full text