Inhibition of angiogenesis and the vital involved pathways are still important challenging questions in the prevention and treatment of many diseases such as cardiovascular disease , corneal neovascularization  and cancer . Despite many efforts in this field, angiogenesis is the leading cause of death in many malignancies. Anti-angiogenic therapeutic strategies are still at the beginning of the path of development and have been associated with severe side effects in many cases . Therefore, emerging novel anti-angiogenic strategies or developing available therapies can be promising for the treatment of many angiogenesis-related diseases.
Cutaneous malignant melanoma is fastest-growing cancer in white populations with a large majority of dermal cancer death. Bevacizumab, a humanized anti-angiogenic immunoglobulin IgG1, is commonly used to inhibit angiogenesis in cutaneous malignant melanoma . Bevacizumab binds to vascular endothelial growth factor (VEGF-A) to block the interaction between VEGF-A and its tyrosine kinase receptor. VEGF-A is a potent stimulant of vascular endothelial cell angiogenesis by activating downstream intracellular signaling pathways leading to proliferation, migration, and sprouting of endothelial cells. Therefore, Bevacizumab offers a potential treatment for angiogenesis-related diseases by inhibiting VEGF-A signaling. However, its systemic administration has been associated with a variety of side effects in many cases . Also, decreased skin absorption, reduced half-life, impaired protein structure, degradation by environmental enzymes, and loss of bioactivity are some of the challenges of local administration of Bevacizumab. To overcome some of these challenges, Bovisizumab encapsulation in nanocarriers has been reported in a variety of studies.
In this study, chitosan polymer-based lipid-coated nanoparticles were designed and synthesized with the aim of local administration of Bevacizumab. The large size of Bevacizumab limits its diffusion transport from NPs. Diffusion transport and release rate is widely associated with the rate of polymer degradation, which is influenced by various factors including the physicochemical characteristics of the polymer, mechanisms related to hydrolysis and enzymatic cleavage of the polymer. Chitosan has attracted a lot of attention in the manufacture of carriers for in vivo drug release, due to its biocompatibility and biodegradability, non-toxicity, high absorption capacity, availability, cost-effectiveness, and antimicrobial and antioxidant properties. In addition, the safety of using chitosan in food and medicine has been approved by the US Food and Drug Administration (FDA).
Here, Lip-Chi nanoparticles were successfully synthesized and used to encapsulate Bevacizumab. In a study by Parisa Badiei et al., the amount of loaded Bevacizumab and the encapsulation efficiency were reported 67% and 15%, respectively . Our results showed an increase in the mentioned parameters which may be due to the change in the exposure time of Bevacizumab with chitosan, increased Bevacizumab concentration, and decreased chitosan concentration . Compared to a study by Sousa F. et al. that used PLGA polymer to load Bevacizumab , our results indicated that chitosan polymer-based nanoencapsulation of Bevacizumab provided a significant increase in the rates of release and dermal absorption of the drug. Also, the amount of Bevacizumab released was greater. Methods to achieve nanoparticles typically include conditions such as suitable pH, high pressure and temperature, use of organic solvents and ionic strength, where any change may lead to structural instability, decreased bioactivity and increased immunogenicity of encapsulated monoclonal antibody. Our results showed that the bioactivity of encapsulated Bevacizumab is maintained in the synthesized Lip-Chi nanoparticles.
Our observations in the study of in vitro and in vivo angiogenesis showed increased suppressor function for Bevacizumab encapsulated in Lip-Chi nanoparticles compared to free Bevacizumab. These nanoparticles appear to provide an efficient environment for maintaining the structure of Bevacizumab against enzymatic degradation and other environmental threatening factors, so that the increased suppressor function of encapsulated Bevacizumab on both in vitro and in vivo angiogenesis may also be due to the release of the drug in greater amount and speed, and may also be due to better preservation of the drug structure until its release and also after its release from Lip-Chi nanoparticles[44, 45]. There are various reports indicating the inhibitory effects of Bevacizumab on angiogenesis using cultured human endothelial cells in vitro. Here, the biological activity of Bevacizumab was determined by its capacity to suppress the endothelial cell proliferation, in vitro and in vivo tubulogenesis. Decreased proliferation and tubulogenesis indicate that Bevacizumab bioactivity was maintained even when encapsulated into Lip-Chi nanoparticles[46, 47]. MTT results also showed that Lip-Chi nanoparticles were not cytotoxic for hBMEC cells, which is consistent with previous reports.
Moreover, the observed increase in dermal absorption of Bevacizumab encapsulated in Lip-Chi nanoparticles may be mediated via lecithin phospholipids. This successful dermal absorption of Bevacizumab can suggest replacing systemic with local administration, which can be an effective strategy to reduce the side effects of systemic administration of Bevacizumab.
Our results suggest that encapsulation of Bevacizumab in chitosan-based lipid-coated nanoparticles is associated with an increased amount and rate of drug release, which may be preferable to other polymers, such as PLGA, where greater drug release is required in a shorter time. Various studies have shown that Bevacizumab encapsulated in PLGA-based nanoparticles exhibits a slow, pH-dependent release profile. Although slow release of the drug may reduce the frequency of administration, it must be proven that the nanoparticle allows the drug to be released even at a slow rate. Our results indicated that the Lip-Chi nanoparticles provide better conditions for more efficient function of Bevacizumab by increasing the efficiency of encapsulation and drug loading as well as dermal cell absorption. The use of these nanoparticles seems to be an effective strategy for local administration of Bevacizumab especially in accessible angiogenesis-related diseases such as cutaneous melanoma.