PLGA nanoparticles for calcitriol delivery

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Two formulation parameters, vitamin/polymer ratio and sonication time, were firstly studied and discussed using cholecalciferol as a drug model. Then, calcitriol- ...

PLGA nanoparticles for calcitriol delivery M J Ramalho, J A Loureiro, B Gomes, M F Frasco, M A N Coelho and M C Pereira LEPABE Departamento de Engenharia Química, Faculdade de Engenharia, Universidade do Porto Rua Dr. Roberto Frias, 4200-465 Porto, Portugal [email protected]

Abstract— Calcitriol, the active metabolite of Vitamin D3, is a potential anticancer agent but exhibits several drawbacks. Therefore, new therapeutic strategies, as specific drug delivery systems, must be developed in order to overcome those limitations. The aim of this work was to prepare poly(lactidecoglycolide) nanoparticles as drug delivery vehicles for calcitriol. Two formulation parameters, vitamin/polymer ratio and sonication time, were firstly studied and discussed using cholecalciferol as a drug model. Then, calcitriol-loaded poly(lactide-coglycolide) nanoparticles with controlled sizes and properties were prepared. The nanoparticles remained stable at storage conditions for several weeks and they were successfully lyophilized to increase their shelf-life using a crioprotectant. In vitro studies using two human cancer cell lines, S2-013 and A549, demonstrated that bare PLGA NPs are biocompatible. Index Terms— Poly(lactic-co-glycolic acid), Calcitriol, 1α,25dihydroxyvitamin D3, Drug Delivery, Nanocarrier, Cancer therapy



Nanoscale drug delivery systems (DDS) using polymeric nanoparticles (NPs) are emerging solutions for the treatment of various diseases. Several polymers have been widely researched as they can effectively deliver drugs to a target site, increasing the therapeutic benefit, while minimizing side effects [1]. Poly (d,l-lactic-co-glycolic acid) (PLGA) is an excellent candidate due to its well established clinical safety. The adjustable biodegradation rate, strength and tunable mechanical properties justify its growing popularity. Also, its special features allow a sustained release and an increased drug bioavailability avoiding the first-pass effect. PLGA NPs are efficiently internalized by targeted cells through an endocytosis mechanism [2], circumventing the multi-drug resistance (MDR) problem [3]. The great success of this biomaterial is testified by the extensive development of its clinical applications. Drugreleasing particles of PLGA were first approved in 1989, with a product named Lupron Depot®. This is a PLGA-based formulation prescribed for the palliative treatment of advanced prostate cancer. There is also a formulation named Telstar® for prostate cancer therapy [4, 5]. In addition, some formulations are currently under clinical trials. A pegylated PLGA NPs formulation is currently ongoing phase II for the delivery of docetaxel in different types of cancer, as prostate and lung cancer [6, 7]; and a PLGA formulation, for the treatment of pain in osteoarthritis of the knee, had already completed phase II of clinical trials [8]. 2015 IEEE 4th Portuguese BioEngineering Meeting Porto, Portugal, 26-28 February 2015

PLGA NPs continue to be a subject of study for a wide range of applications, from food industry [9] to medicine. PLGA NPs can be used as promising diagnosis tools [10] and as devices for the delivery of several drugs. Recently, Jain and colleagues (2012) developed pegylated NPs coupled with folate as vehicles for the oral delivery of insulin. In vivo studies with adult male rats demonstrated that NPs increased oral bioavailability, comparatively to subcutaneously administered insulin solution [11]. In another work, Jose and team (2014) presented surface modified PLGA nanoparticles for brain delivery of Bacoside-A, a neuroprotective drug, for the treatment of neurodegenerative disorders. The NPs were coated with polysorbate 80 to enhance blood brain barrier (BBB) permeability’s. In vivo studies with albino rats successfully demonstrated the potential of these nanoparticles as vehicles for brain targeting [12]. Despite its wide range of applications, PLGA may provide a major benefit in cancer therapy research. PLGA NPs could offer a suitable solution taking advantage of a selective drug delivery to tumor tissue either by passive targeting with the enhanced permeability and retention effect (EPR) [3] or by active targeting using functionalized NPs [1]. Indeed, several recent studies have revealed promising results for cancer therapy. Martin and colleagues (2013) prepared PLGA NPs coated with positively charged chitosan. Its mucoadhesive properties enhanced binding and internalization of the PLGA NPs allowing an efficient delivery of survivin small-interfering RNA (siRNA). Survivin is an apoptosis inhibitor overexpressed in bladder cancer cells. In vitro and in vivo studies showed a decrease in survivin expression, proving that this system is an efficient vehicle for bladder cancer treatment [13]. Zhu and co-workers (2014) developed PLGA NPs for the codelivery of docetaxel and vitamin E TPGS (d-alpha tocopheryl polyethylene glycol 1000 succinate). The latter was used as a bioactive agent to inhibit P-glycoprotein to overcome MDR in cancer cells. In vivo and in vitro studies proved that this strategy to overcome MDR problem was able to improve docetaxel antineoplastic activity [14]. In this work, we propose PLGA NPs for the delivery of Vitamin D3. Vitamin D3, a well-known regulator of calcium homeostasis and bone mineralization, is recently being associated with many additional functions including antineoplastic activity [15]. Several pathways by which calcitriol, the active metabolite of Vitamin D3, may prevent, treat or stop tumor growth have been described [16, 17]. Despite calcitriol’s multiple medicinal benefits, its low

bioavailability due to as its short half-life in bloodstream [18] and first-pass effect [19], and its high toxicity continue to be highlighted as major challenges in developing formulations for clinical use. Its anti-tumoral activity only occurs at supraphysiological doses associated with a high risk of hypercalcemia [20]. Calcitriol is also sensitive to many external and environmental factors that may affect its molecular structure and therefore its activity [21]. These limitations justify the need of designing and developing an ideal formulation for calcitriol delivery to cancer cells. This work aims to address the need to design a suitable nanosystem for caclitriol delivery able to overcome its limitations, through its entrapment in PLGA NPs. II.


The main goal of the present work is to develop a suitable PLGA-based nanocarrier and to evaluate its capacity to encapsulate and to deliver calcitriol, improving its therapeutic efficiency. III.


This work is being developed at the Laboratory for Process Engineering, Environmental, Biotechnology and Energy (LEPABE) at the Faculty of Engineering of University of Porto in collaboration with Institute of Molecular Pathology and Immunology of University of Porto (IPATIMUP). IV.


The overall research work comprises four main tasks: (A) preparation of PLGA NPs and evaluation of the effect of different experimental parameters on NPs properties; (B) encapsulation of Vitamin D3 active metabolite and the physicochemical characterization of the prepared nanosystems; (C) evaluation of the stability of the prepared nanocarrier at storage conditions and after freeze-drying process; (D) study of the calcitriol-loaded PLGA NPs effects on human cancer cell lines. A.

PLGA NPs preparation and assessment of the influence of experimental parameters on NPs properties PLGA NPs were prepared using the emulsion-solvent evaporation technique. For that purpose, PLGA (10 mg) was dissolved in ethyl acetate, and for encapsulation vitamin (1 mg or 0.1 mg) was added to this organic phase. An aqueous solution of 1% (w/v) pluronic®F127 was added dropwise to the organic phase. Then, the solution was vortexed and emulsified by sonication at an ultrasonic frequency of 45 kHz. The emulsion was subsequently poured into a solution of 0.1% (w/v) pluronic®F127 and stirred at room temperature until complete evaporation of the organic solvent. The resulting suspension was filtered (0.2 μm, Millex-GP Filter Units, Merck Millipore, Germany), and incubated at 4 ºC overnight to increase NPs stability.

drug model in this step. After defining the optimal conditions, PLGA NPs were prepared for calcitriol entrapment. B.

PLGA NPs physicochemical characterization The size, polydispersity index (PdI), zeta potential, morphology, loading capacity, encapsulation efficiency and in vitro release profile were the parameters used to characterize the produced nanoparticles. Size distribution and zeta potential were determined by Dynamic Light Scattering (DLS) and Electrophoretic Light Scattering (ELS), respectively. Morphological analysis was conducted by Transmission Electron Microscopy (TEM). The in vitro release behavior of calcitriol entrapped in the PLGA NPs was evaluated in PBS (0.01 M, pH 7.4 at 37 °C) over seven days. C.

Nanoparticles stability studies In order to evaluate the nanosystem stability, the prepared PLGA NPs were stored at 4 °C and modifications in their size and zeta potential were assessed over a period of nine weeks by DLS and ELS measurements, respectively. DLS and ELS analysis were also performed to assess the occurrence of aggregation or modification of the PLGA NPs properties after freeze-drying. Lyophilization was carried out in a BenchTopTM K series freeze-dryer (VirTis,NY, USA) at 5 x 10-5 bar and –95 ºC for 48 h. The effect of sucrose, at the concentration of 1% (w/v), as a cryoprotective agent on the NPs stability during lyophilization was also determined. D.

Cellular studies In vitro cell assays were performed with different human cancer cell lines. It was used S2-013, a well differentiated tubular adenocarcinoma and moderately metastatic subline cloned from the human pancreatic tumor cell line SUIT-2 [22] and A549, a human non-small cell lung carcinoma line [23]. Laser Scanning Confocal Microscopy (LSCM) was used to visualize the PLGA uptake by cells and cytotoxicity was determined by Sulforhodamine B (SRB) assay. Cell cycle analysis by quantitation of DNA content in PI-stained cells was performed using flow cytometry (FCM). This task is under progress with promising preliminary results presented below. V.



Physicochemical features of PLGA nanoparticles PLGA NPs were prepared by a single emulsion solvent evaporation method and the influence of two experimental parameters on the properties of the NPs was evaluated. The attained results are presented in tables I and II. TABLE I.


Two experimental parameters, the sonication step and the vitamin/polymer ratio, were tested in order to achieve the adequate experimental conditions. Due to the cost of calcitriol, cholecalciferol, the inactive form of vitamin D3, was used as a.

Sonication time

Mean size (nm) a

PdI a

Zeta Potential (mV) a

1 min

176 ± 2

0.096 ± 0.090

- 34 ± 4

5 min

174 ± 4

0.041 ± 0.003

- 40 ± 1

10 min

173 ± 18

0.054 ± 0.027

- 36 ± 3

Data represented as mean ± SD (n=3)

TABLE II. PLGA NPs Cholecalciferolloaded Calcitriol-loaded b.


Mean size (nm) b

Pd b

Zeta potential (mV) b

LE (%)b

LC (%)b

172 ± 8

0.069 ± 0.033

- 33 ± 1

81 ± 17

0.8 ± 0.1


189 ± 7

0.089 ± 0.060

- 30 ± 6

83 ± 2

8.3 ± 0.2


187 ± 2

0.038 ± 0.021

- 33 ± 5

57 ± 8

5.7 ± 0.9

Data represented as mean ± SD (n=3)

Since sonication is a crucial step to define the properties of the nanoparticles [24], the influence of the duration of this step on the features of the produced unloaded PLGA NPs was assessed. As table I shows, the unloaded NPs, produced with three different sonication times, exhibited mean sizes varying between 173 and 176 nm. Increasing sonication time, didn’t significantly alter NPs mean size (p>0.05). Also, no significant changes were verified (p>0.05) for PdI and zeta potential values. The sonication time selected for the following experiments was 5 minutes. The single emulsion solvent evaporation method also allowed the encapsulation of vitamin D3 in the PLGA NPs. Different vitamin/polymer ratios were used to verify its influence on NPs properties. The attained results for mean size, PdI, zeta potential, loading capacity and encapsulation efficiency values for the PLGA NPs loaded with cholecalciferol and calcitriol are shown in table II. As it is shown in tables I and II, the size of 10% vitaminloaded NPs (189 ± 7 nm for cholecalciferol-loaded, and 187 ± 2 for calcitriol-loaded, respectively) increased significantly (p0.05). Table 2 also shows the significant increase (p0.05). The attained results for encapsulation efficiency (EE) for both encapsulated forms of Vitamin D3 are also expressed in percentages in table II. No significant changes of EE values (p>0.05) were observed for the different cholecalciferol/polymer ratios reported. However for 10% ratios, the obtained results significantly decrease (p

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