The Antitumor Effect of Topotecan Loaded Thiolated Chitosan-Dextran Nanoparticles for Intravitreal Chemotherapy: A Xenograft Retinoblastoma Model.

Purpose: This research intended to fabricate the thiolated chitosan-dextran nanoparticles (NPs) containing topotecan (TPH-CMD-TCS-NPs) to assess the ability of NPs in improving the efficacy of intravitreal chemotherapy of retinoblastoma in a rabbit xenograft model. Methods: The coacervation process was used to produce the NPs. The cellular uptake of Cyanine-3 (CY3)-labeled NPs were investigated in human retinoblastoma Y79 cells using confocal microscopy. Also, the prepared TPH-CMD-TCS-NPs were tested in vitro by the tetrazolium dyes II (XTT) and flow cytometry in order to assess their cytotoxicity. In addition, a rabbit xenograft model of retinoblastoma was developed to test the antitumor effectiveness of TPH-CMD-TCS-NPs through intravitreal administration. Results: NPs had a mean diameter, polydispersity index, and zeta potential of 30 ± 4 nm, 0.24 ± 0.03 and +10 ± 3 mV, respectively. NPs (IC50s 40.40 compared to 126.20 nM, P = 0.022) were more effective than free topotecan as a dose-based feature. The tumor reaction to intravitreal chemotherapy with NPs was measured by evaluating the percentage of necrosis in the tumor tissue (91 ± 2%) and vitreous seeds (89 ± 9%) through hematoxylin and eosin (H&E) staining. In comparison with the control group, the TPH-CMD-TCs-NPs treated group showed a significant decrease in tumor volume seven days after the intravitreal injection (P = 0.039). No significant changes were found in the ERG parameters after the intravitreal injection of TPH-CMD-TCs-NPs or TPH (P > 0.05). Conclusion: This investigation revealed definitive antitumor efficacy of TPH-CMD-TCS-NPs by intravitreal administration in the rabbit xenograft retinoblastoma model.

Erratum: Biodistribution of Cy5-labeled Thiolated and Methylated Chitosan-Carboxymethyl Dextran Nanoparticles in an Animal Model of Retinoblastoma.

[This corrects the article DOI: 10.18502/jovr.v17i1.10171.].

Biodistribution of Cy5-labeled Thiolated and Methylated Chitosan-Carboxymethyl Dextran Nanoparticles in an Animal Model of Retinoblastoma.

PURPOSE: The use of more potent medicine for local chemotherapy of retinoblastoma in order to minimize local and systemic adverse effects is essential. The main goal of this investigation was to assess the biodistribution of thiolated and methylated chitosan-carboxymethyl dextran nanoparticles (CMD-TCs-NPs and CMD-TMC-NPs) following intravitreal (IVT) injection into rat eyes with retinoblastoma. METHODS: An ionic gelation method was used to fabricate Cy5-labelled CMD-TCs-NPs and CMD-TMC-NPs. The NPs were characterized. Cellular internalization of Cy5-labelled NPs was investigated using confocal microscopy and the absorption of labeled NPs was quantified by flow cytometry in human retinoblastoma (Y79) cells. In addition, the Cy5-labeled distribution of nanoparticles in the posterior segment of the eye was histologically imaged by confocal microscopy after IVT injection of NPs into the eyes of rats with retinoblastoma. RESULTS: CMD-TCs-NPs and CMD-TMC-NPs showed a mean diameter of 34 ± 3.78 nm and 42 ± 4.23 nm and zeta potential of +11 ± 2.27 mV and +29 ± 4.31mV, respectively. The in vivo study of intraocular biodistribution of Cy5-labeled CMD-TCs-NPs and CMD-TMC-NPs revealed that there is more affinity of CMD-TCs-NPs to the retina and retinoblastoma tumor after IVT administration while methylated chitosan nanoparticles are immobilized in the vitreous and are not able to reach the retina even after 24 hr. CONCLUSION: The ionic gelation technique was efficient in synthesizing a biocompatible polymeric nanosystem for drug delivery into the posterior segment of the eye. The current study demonstrated increased ocular bioavailability of CMD-TCs-NPs relative to CMD-TMC-NPs in retinoblastoma induced rat eyes.

Efficacy of topotecan nanoparticles for intravitreal chemotherapy of retinoblastoma.

Retinoblastoma (Rb) is the most common intraocular malignancy in children that accounts for approximately 4% of all pediatric malignancies. Since chemotherapy is a widely practiced treatment for Rb, there is a growing interest in developing new and effective drugs to overcome systemic and local side effects of chemotherapy to improve the quality of life and increase the chances of survival. This study sought to fabricate thiolated chitosan nanoparticles containing topotecan (TPH-TCs-NPs) with a view of enhancing drug loading and release control. This research was also designed to assess the ability of TPH-TCs-NPs to improve cell association, increase treatment efficacy in retinoblastoma cells and xenograft-rat-model of retinoblastoma, and overcome current topotecan hydrochloride (TPH) intravitreal administration challenges, including stability loss and poor cellular uptake. Modified ionic gelation method was optimized to fabricate TPH-TCs-NPs and TPH-TMC-NPs (N-trimethyl chitosan nanoparticles containing TPH). We characterized the NPs and quantified topotecan loading and release against a free TPH standard. The efficacy of TPH-NPs was quantified in human retinoblastoma cells (Y79) by XTT and flow cytometry measurement. In addition, Y79 cells were injected intravitreally in both eyes of immunodeficient wistar albino rats to create a xenograft-rat-model to compare the antitumor effectiveness of TPH-NPs and TPH by intravitreal administration. TPH-NPs complexation was confirmed by EDX, FTIR, and DSC techniques. TPH-TCs-NPs and TPH-TMC-NPs had high encapsulation efficiency (85.23 ± 2 and 73.34 ± 2% respectively). TPH-TCs-NPs showed a mean diameter, polidispersity index, and zeta potential of 25±2 nm, 0.21 ± 0.03 and +12 ± 2 mV, respectively. As a function of dose, TCs and TMC NPs were more efficacious than free topotecan (IC50s 53.17 and 85.88 nM, relative to 138.30 nM respectively, P = 0.012). Kruskal-Wallis test showed a statistically significant difference between the groups. Additionally, a significant difference between the tumor control and TPH-TCs-NPs treated group in xenograft-rat-model ( Range of P-value: 0.026 to 0.035) was shown by Bonferroni post hoc test. The current investigation demonstrated enhanced efficacy and association of TPH-TCs-NPs relative to free TPH in retinoblastoma cells and tumor in vitro and in vivo.

Safety of Intravitreal Injection of Stivant, a Biosimilar to Bevacizumab, in Rabbit Eyes.

PURPOSE: To evaluate the safety of intravitreal injection of Stivant, a biosimilar to bevacizumab, in rabbits using electrophysiological and histological analysis. METHODS: Both eyes of 41 New Zealand albino rabbits were injected with 0.1 mL (2.5 mg) of Stivant. The rabbits were scheduled to be sacrificed 1, 2, 7, 14, and 28 days after injection for histopathological evaluations. Clinical examinations and electroretinography (ERG) were performed at baseline and just before sacrificing the rabbits. Fourteen separate rabbits received a reference drug (Avastin) and were considered as the control group. Furthermore, three other rabbits received the same volume of saline (saline control group). Rabbits of both control groups were sacrificed four weeks after injection. ERG was performed 1, 2, 7, 14, and 28 days after injections. RESULTS: No significant difference was observed in a- and b-wave amplitudes and latency after intravitreal Stivant injection between baseline and different time points. Moreover, there was no statistically significant difference in wave amplitudes and latency between the Stivant and control groups. The histology of rabbit eyes of the Stivant and control groups after intravitreal injections was not distinguishable. CONCLUSION: The biosimilar Stivant, up to a dose of 2.5 mg, did not appear to be toxic to the retina in albino rabbits. These results suggest that this drug could be a safe and inexpensive alternative to intravitreal bevacizumab. The efficacy of these injections was not investigated in this study and needs to be evaluated in future studies.

Safety of Intravitreal Injection of Biosimilar of Aflibercept in Rabbit Eyes.

PURPOSE: To assess the safety of biosimilar intravitreal aflibercept (CinnaGen Co., Iran) compared to the reference product (Eylea®; Bayer Schweiz AG, Zurich, Switzerland) in rabbit eyes through functional and histologic studies. METHODS: Forty New Zealand albino rabbits were recruited to the study and were divided into four groups to be sacrificed at 48 hours, one, two, and four weeks after injections. In each group, five rabbits received 0.05 mL (2 mg) biosimilar aflibercept in the right eye and 0.05 mL saline in the left eye as the control, and in a similar manner, the remaining five rabbits received the reference drug in the right eye and saline in the left eye. All the rabbits underwent comprehensive ophthalmic examination and electroretinography (ERG) tests at baseline and also just before enucleation at the specific predefined time points. The enucleated eyes were prepared for retinal toxicity histological examination. RESULTS: No retinal toxicity was observed based on histologic and ERG findings in all groups. Choroidal congestion was revealed after 1 week in an eye that was injected with biosimilar aflibercept, although the similar finding was detected in the contralateral eye which received saline. Also, one subject which received the reference drug showed chronic vitritis and lymphoplasmocytic reaction of the optic disc at week 4. The remaining subjects showed no histologic changes. CONCLUSION: The 2 mg intravitreal injection of biosimilar aflibercept (CinnaGen Co., Iran) was found to be nontoxic in rabbit eyes in the short-term period. Further studies are required to warrant the efficacy and safety profile of the drug in human subjects.

Safety and Bioavailability of Complete and Half-Dose Intravitreal Ziv-Aflibercept in an Experimental Model: Contralateral Eye Study.

BACKGROUND AND OBJECTIVE: To evaluate the safety and bioavailability of complete and half-dose of intravitreal ziv-aflibercept (IVZ) in an experimental model. MATERIALS AND METHODS: Thirty-two eyes of 16 male rabbits received one IVZ injection under anesthesia and the operating microscope. All right and left eyes received 1,250 µg/0.05 mL and 625 µg/0.05 mL of ziv-aflibercept, respectively. Then, rabbits were randomly allocated to four groups (four rabbits in each group). The rabbits were euthanized at predesignated intervals (at 24, 168, 336, and 720 hours), and the eyes were enucleated. Indirect ophthalmoscopy, vitreous sampling, and electrophysiological recordings were obtained before euthanization. Histological examination was performed after enucleation. Vitreous samples were evaluated by enzyme-linked immunosorbent assay to measure the concentration of aflibercept. RESULTS: No serious drug-related ocular inflammation and toxicity or systemic adverse events were identified. Electroretinogram findings showed no significant difference to the baseline measurements. Remaining vitreal concentrations of ziv-aflibercept injection for the 625 µg/mL group were 416 µg/mL, 349 µg/mL, 124 µg/mL, 41.2 µg/mL, and 18.1 µg/mL (± 10 µg/mL) and for the 1,250 µg/mL group were 833 µg/mL, 737 µg/mL, 284 µg/mL, 87.3 µg/mL, and 38.2 µg/mL (± 10 µg/mL), at zero, 24, 168, 336, and 720 hours after injection, respectively. The vitreous concentration of aflibercept was analyzed by one-compartment model. The area under curve from time 0 to the end point (AUC last) was 147,637 hours × µg/mL for the complete dose group (1,250 µg/0.05mL) and 68,498 hours × µg/mL for the half-dose group (625 µg/0.05 mL). The assessed vitreous half-life of ziv-aflibercept was 113 hours in both groups. CONCLUSIONS: IVZ proved to be safe and well tolerated, even in the complete dose group. It seems to be a cost-effective therapeutic option for the treatment of retinal vascular diseases. However, the long-term safety and efficacy of intravitreal ziv-aflibercept remain unknown. [Ophthalmic Surg Lasers Imaging Retina. 2019;50:785-790.].

Alginate Microcapsules as Nutrient Suppliers: An In Vitro Study.

OBJECTIVES: Alginate, known as a group of anionic polysaccharides extracted from seaweeds, has attracted the attention of researchers because of its biocompatibility and degradability properties. Alginate has shown beneficial effects on wound healing as it has similar function as extracellular matrix. Alginate microcapsules (AM) that are used in tissue engineering as well as Dulbecco's modified Eagle's medium (DMEM) contain nutrients required for cell viability. The purpose of this research was introducing AM in medium and nutrient reagent cells and making a comparison with control group cells that have been normally cultured in long term. MATERIALS AND METHODS: In this experimental study, AM were shaped in distilled water, it was dropped at 5 mL/hours through a flat 25G5/8 sterile needle into a crosslinking bath containing 0.1 M calcium chloride to produce calcium alginate microspheres. Then, the size of microcapsules (300-350 µm) were confirmed by Scanning Electron Microscopy (SEM) images after the filtration for selection of the best size. Next, DMEM was injected into AM. Afterward, adiposederived mesenchymal stem cells (ADSCs) and Ringer's serum were added. Then, MTT and DAPI assays were used for cell viability and nucleus staining, respectively. Also, morphology of microcapsules was determined under invert microscopy. RESULTS: Evaluation of the cells performed for spatial media/microcapsules at the volume of 40 µl, showed ADSCs after 1-day cell culture. Also, MTT assay results showed a significant difference in the viability of sustained-release media injected to microcapsules (P<0.05). DAPI staining revealed living cells on the microcapsules after 1 to 7-day cell culture. CONCLUSIONS: According to the results, AM had a positive effect on cell viability in scaffolds and tissue engineering and provide nutrients needed in cell therapy.

The study of CaO and MgO heterogenic nano-catalyst coupling on transesterification reaction efficacy in the production of biodiesel from recycled cooking oil.

BACKGROUND: Fossil fuels' pollution and their non-renewability have motivated the search for alternative fuels. Some common example of seed oils are sunflower oil, date seed oil, soy bean oil. For instance, soy methyl and soy-based biodiesel are the main biodiesel. Biodiesel is a clean diesel fuel that can be produced through transesterification reaction. Recycled cooking oil, on the other hand, is one of the inexpensive, easily available sources for producing biodiesel. RESULTS: This article is aimed at production of biodiesel via trans-esterification method, Nano CaO synthesis using sol-gel method, and Nano MgO synthesis using sol-gel self-combustion. Two catalysts' combination affecting the reaction's efficacy was also discussed. Optimum conditions for the reaction in the presence of Nano CaO are 1.5 % weight fracture, 1:7 alcohol to oil proportion and 6 h in which biodiesel and glycerin (the byproduct) are produced. Moreover, the optimum conditions for this reaction in the presence of Nano CaO and Nano MgO mixture are 3 % weight fracture (0.7 g of Nano CaO and 0.5 g of Nano MgO), 1:7 alcohols to oil proportion and 6 h. CONCLUSIONS: Nano MgO is not capable of catalyzing the transesterification by itself, because it has a much weaker basic affinity but when used with Nano CaO due to its surface structure, the basic properties increase and it becomes a proper base for the catalyst so that CaO contact surface increases and transesterification reaction yield significantly increases as well. This study investigates the repeatability of transesterification reaction in the presence of these Nano catalysts as well.

Prophylactic effect of topical silica nanoparticles as a novel antineovascularization agent for inhibiting corneal neovascularization following chemical burn.

BACKGROUND: Angiogenesis-related corneal blindness includes the spectrum of corneal diseases that are caused by pathological angiogenesis, leading to untoward visual impairment. The purpose of this study was to investigate the antineovascularization effect of topical silica nanoparticles (SiNPs) in inhibiting chemical-burn-induced corneal neovascularization. MATERIALS AND METHODS: A total number of 20 corneas of 10 Wistar Albino rats were included in this study. Silver nitrate cauterization was pressed to the central cornea for 5 s to induce corneal neovascularization. They were randomly allocated to case and control groups (ten eyes in each group). SiNPs were synthesized by the reverse microemulsion method. SiNPs drop 1 mg/ml was started in ten eyes and artificial tear drop was started in the control group (ten eyes) immediately after chemical cauterization. Video-based photography was performed before and after treatment. Corneal image analysis was performed on each cornea using an image analysis software program. All rats were euthanized and the eyes were sent for histopathologic examinations14 days after chemical cauterization. RESULTS: Scanning electron microscopy (SEM) images showed spherical-shaped particles. The mean size and polydispersity index of prepared SiNPs were 30.1 ± 5.6 nm and 0.254 ± 0.11, respectively. Fourteen days after chemical cauterization, the mean vascularized corneal area was 21% of total corneal area in the case group and 85% in the control group (P < 0.05). The control group revealed more extensive intrastromal vascularization compared with the case group in histopathologic examinations (P < 0.05). CONCLUSIONS: SiNPs is an effective modality for inhibiting corneal neovascularization following chemical burn in an experimental model. Further investigations are suggested for evaluation of its safety and efficacy in human eyes.

Penetration of silicate nanoparticles into the corneal stroma and intraocular fluids.

PURPOSE: The aim of this study was to investigate transmission of topical silicate nanoparticles (SiNPs) through the corneal stroma, anterior chamber, and vitreous fluids by scanning electron microscopy (SEM), transmission electron microscopy (TEM), and inductively coupled plasma atomic emission spectrometry (ICP-AES), respectively. METHODS: SiNPs with a mean diameter of 40.6 ± 5.6 nm determined by dynamic light scattering were used in this study. The permeability of SiNPs was examined across isolated corneal buttons over a 30-minute period. To visualize the transport and diffusion of nanoparticles through the corneal tissue, SiNPs were applied over the corneal surface and evaluated at 5 and 30 minutes after SiNPs loading for SEM and 15 minutes for TEM. Sections of 10-µm thickness were cut and visualized using SEM. TEM study was performed on 70- to 90-nm-thick sections. ICP-AES was used to determine the concentration of SiNPs. RESULTS: The determined range of synthesized SiNPs by dynamic light scattering was 40 nm (41.9 ± 5.6 nm). Transmission of SiNPs through the corneal stroma was shown successfully with electron microscopic (SEM and TEM) images. The ICP-AES results revealed SiNPs in the anterior chamber and vitreous fluid. CONCLUSIONS: Topical administration of SiNPs, as a noninvasive, and available modality with acceptable penetration through the corneal stroma and deep into the intraocular fluids including the anterior chamber and vitreous cavity, may be considered as a suitable alternative to invasive intravitreal injection of other expensive antineovascularization agents.