Elsevier

Biomedicine & Pharmacotherapy

Volume 92, August 2017, Pages 394-402
Biomedicine & Pharmacotherapy

Original article
Antiangiogenic activity of PLGA-Lupeol implants for potential intravitreal applications

https://doi.org/10.1016/j.biopha.2017.05.093Get rights and content

Abstract

Uncontrolled angiogenesis is directly associated with ocular diseases such as macular degeneration and diabetic retinopathy. Implantable polymeric drug delivery systems have been proposed for intravitreal applications and in the present work, we evaluated the antiangiogenic potential of PLGA ocular implants loaded with the triterpene lupeol using in vitro and in vivo models. The drug/polymer physiochemical properties of the lupeol-loaded PLGA were validated as functionally similar using differential scanning calorimetry, Fourier transform infrared spectroscopy, and scanning electron microscopy. Interestingly, in an in vitro culture system, lupeol (100 μg/mL and 250 μg/mL) was capable to inhibited the proliferation as well as the migration of Human Umbilical Vein Endothelial Cells (HUVEC), without interfering in cell viability, promoting a significant reduction in the percentage of vessels (39.41% and 44.12%, respectively), compared with the control group. In vivo test, by using the chorioallantoic membrane (CAM) model, lupeol-loaded PLGA ocular implants showed antiangiogenic activity comparable to the FDA-approved anti-VEGF antibody Bevacizumab. Overall, our results suggest lupeol-loaded PLGA ocular implants were able to inhibit the angiogenic process by impairing both proliferation and migration of endothelial cells.

Introduction

Angiogenesis is a natural process that occurs in the human body during fetal development or as a response to tissue damage, as part of wound healing process and renewing the blood flux in damage areas [1], [2]. When the human body loses its capacity to maintain adequately the equilibrium of angiogenic mediators, some diseases can develop, including arthritis, cancer, endometriosis, psoriasis, macular degeneration related to age and proliferative diabetic retinopathy [3], [4]. Macular degeneration related to age is the main cause of blindness in persons older than 60 years in industrialized countries [5]. The prevalence of blindness can vary between 10 and 15% among affected persons and estimates have shown a significantly rates increase until 2030 [6]. Currently, available treatments are limited to palliatives such as photodynamic therapy, intravitreal injections with corticoids, antiangiogenic compounds applied directly into eye and laser photocoagulation [7], [8], [9], [10]. The proliferative diabetic retinopathy is characterized by new important vascularization in retina, following to intravitreal interface, with possible loses of normal visual characteristics mainly due to traditional retinal detachment. In this sense, the inhibition of angiogenesis can be considered an important strategy to treat these intraocular diseases and have been explored in many published works [11], [12], [13], [14].

Lupeol is a natural pentaciclic triterpene with a lupane scaffold that can be found in diverse vegetables, including white cabbage, red pepper, cucumber, tomatoes, carrots, peas, and soy. Moreover, administration of lupeol does not result in systemic toxicity in animal models in doses ranging from 30 to 2000 mg kg−1 [15]. Triterpene can be isolated from medicinal plants, i.e. Celastraceae family plants, displaying clinically relevant biological properties related to inflammation, arthritis, cardiovascular disorders, cancer and wound healing processes [16], [17], [18]. You et al. [19] evaluated the antiangiogenic activity of lupeol on a tube-like formation assay using HUVEC cells (Human umbilical vein endothelial cells). The results revealed lupeol capacity to inhibit 80% of angiogenic processes at a non-toxic dose of 50 μg mL−1, whereas lower doses showed a significant reduction in antiangiogenic activity reaching only 40% of initial values. Other studies using an endothelial cell model have indicated that triterpenes can modulate growth factors such VEGF (Vascular endothelial growth factor) and are capable of inducing cellular differentiation, seeking to inhibit the vascular tissue growth, displaying an important antiangiogenic effect [20], [21], [22], [23].

Due to anatomical and physiologic characteristics of the eye, administration of ophthalmic medicines is difficult and many studies showed that only approximately 5% of the administrated dose are absorbed by intraocular tissues, making the treatment unfeasible for diseases located in posterior segment of the eye. Other available treatments require the use of high drug doses or are too invasive as intravitreal injections, exhibiting great risks and potentially serious side effects to the patient [24], [25], [26]. Seeking to overcome this negative scenario, research has been dedicated to developing new drug delivery systems, such as polymeric implants with the overall goal to be more selective and achieve favorable bioavailability profiles through sustained releasing of the therapeutic cargo [27], [28]. Such systems offer many advantages, including favorable patient compliance, biocompatibility, predictable biodegradation kinetic and mechanical resistance in various intravitreal applications [29], [30], [31]. In order to mitigate the cumulative risks associated with repeated intravitreal injections, some implantable polymeric systems have been approved by FDA and currently available, for example: Ozurdex® (Dexamethasone Intravitreal Implant); Iluvien® (Fluocinolone acetonide intravitreal implant) and Triesence® (Triamcinolone acetonide). In these systems, any cytotoxicity was observed as well as significant antiangiogenic activity were obtained, displaying the applicability of these systems in intravitreal applications. However, at present, no steroid has achieved US FDA approval for the treatment of pathologies associated to angiogenesis (except for ranibizumab, a monoclonal antibody).

In this work, we aimed to evaluate the anti-angiogenesis activity of PLGA ocular implants containing the lupeol, a non-steroid compound, in both in vitro and in vivo models.

Section snippets

Chemical and reagents

Poly (d,l-lactide-co-glycolide) in ratio of 75:25 [PLGA (75:25)] was purchased from Boehringer Ingelheim (Germany). All the solvents and reagents used in buffer solutions, in the preparation of the implants, and mobile phase were HPLC or analytical grade. Water was distilled, deionized and filtered through a 0.22 μm filter (Millipore, USA).

Lupeol extraction

Dried and pulverized stem of Maytenus salicifolia (2525.9 g) were subjected to exhaustive maceration in n-hexane at room temperature, yielding 14.7 g of hexane

Statistical analysis

The mean values and standard deviation were calculated. The statistical parameters were analyzed through ANOVA followed post-test of Tukey or Bonferroni where p  0.05 was considered as statistically significant. The Mann–Whitney non-parametric test was used to compare outcomes in both groups. The unpaired-test was used to compare outcomes of percent blood vessels in the CAM study. Values of p  0.05 were considered to be statistically significant. Interrelation between dark-adapted b-wave

Lupeol characterization

Lupeol was obtained as a white solid and its molecular structure are presented in Fig. 1. The IR spectrum of Lupeol presented absorption bands at 3313 cm−1 (single bondOH), 1638 (Cdouble bondC) and 878 (double bondCH). The 13C NMR spectral data were similar to the one related by Kundu [34].

Differential scanning calorimetry (DSC)

DSC curves for lupeol, blank PLGA 75:25 and lupeol-loaded PLGA ocular implants are presented in Fig. 2 (respectively A–C). Lupeol presented during the heating two important peaks that were correlated to phase transitions and melting point.

Conclusion

Polymeric implants constituted by PLGA polymer and lupeol at 30% (w/w) were prepared and physicochemical characterized, revealing existing only physical interactions among its contents. These findings demonstrate that lupeol molecular structure was maintained unaltered in polymeric matrix and through normal biodegradation can be released without modifications. Although lupeol presents antitumor and antiangiogenic effects in different models, it is clear these effects do not necessarily derive

Acknowlegdments

The authors would like to thank CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) and FAPEMIG (Fundação de Amparo à Pesquisa do Estado de Minas Gerais) for their financial support.

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