Encapsulated Cell Technology for the Delivery of Biologics to the Mouse Eye.

Many current therapeutics under development for diseases of the posterior pole of the eye are biologics. These drugs need to be administered frequently, typically via intravitreal injections. Encapsulated cells expressing the biologic of choice are becoming a tool for local protein production and release (e.g., via long-term drug delivery). In addition, encapsulation systems utilize permeable materials that allow diffusion of nutrients, waste, and therapeutic factors into and out of cells. This occurs while masking the cells from the host immune response, avoiding the need for suppression of the host immune system. This protocol describes the use of alginate as a polymer in microencapsulation coupled with the electrospray method as a microencapsulation technique. ARPE-19 cells, a spontaneously arising human RPE cell line, has been used in long-term cell therapy experiments due to its lifetime functionality, and it is used here for encapsulation and delivery of the capsules to mouse eyes. The manuscript summarizes the steps for cell microencapsulation, quality control, and ocular delivery.

A Novel Alginate-Based Delivery System for the Prevention and Treatment of Pressure-Overload Induced Heart Failure.

Background: α-CGRP (alpha-calcitonin gene related peptide) is a cardioprotective neuropeptide. Our recent study demonstrated that the administration of native α-CGRP, using osmotic mini-pumps, protected against transverse aortic constriction (TAC) pressure-induced heart failure in mice. However, the short half-life of peptides and the non-applicability of osmotic pumps in humans limits the use of α-CGRP as a therapeutic agent for heart failure (HF). Here, we sought to comprehensively study a novel α-CGRP delivery system using alginate microcapsules to determine its bioavailability in vivo and to test for cardioprotective effects in HF mice. Methods: Native α-CGRP filled alginate microcapsules (200 µm diameter) were prepared using an electrospray method. The prepared alginate-α-CGRP microcapsules were incubated with rat cardiac H9c2 cells, mouse cardiac HL-1 cells, and human umbilical vein endothelial cells (HUVECs), and the cytotoxicity of the alginate-α-CGRP microcapsules was measured by a trypan-blue cell viability assay and a calcium dye fluorescent based assay. The efficacy of the alginate-α-CGRP microcapsules was tested in a TAC-pressure overload mouse model of heart failure. Male C57BL6 mice were divided into four groups: sham, sham-alginate-α-CGRP, TAC-only, and TAC-alginate-α-CGRP, and the TAC procedure was performed in the TAC-only and TAC-alginate-α-CGRP groups of mice to induce pressure-overload heart failure. After 2 or 15 days post-TAC, alginate-α-CGRP microcapsules (containing an α-CGRP dose of 6 mg/kg/mouse) were administered subcutaneously on alternate days, for 28 days, and echocardiography was performed weekly. After 28 days of peptide delivery, the mice were sacrificed and their hearts were collected for histological and biochemical analyses. Results: Our in vitro cell culture assays showed that alginate-α-CGRP microcapsules did not affect the viability of the cell lines tested. The alginate-α-CGRP microcapsules released their peptides for an extended period of time. Our echocardiography, biochemical, and histology data from HF mice demonstrated that the administration of alginate-α-CGRP microcapsules significantly improved all cardiac parameters examined in TAC-mice. When compared to sham mice, TAC significantly decreased cardiac functions (as determined by fraction shortening and ejection fraction) and markedly increased heart and lung weight, left ventricle (LV) cardiac cell size, cardiac apoptosis, and oxidative stress. In contrast, the administration of alginate-α-CGRP microcapsules significantly attenuated the increased heart and lung weight, LV cardiac cell size, apoptosis, and oxidative stress in TAC mice. Conclusion: Our results demonstrate that the encapsulation of α-CGRP in an alginate polymer is an effective strategy to improve peptide bioavailability in plasma and increase the duration of the therapeutic effect of the peptide throughout the treatment period. Furthermore, alginate mediates α-CGRP delivery, either prior to the onset or after the initiation of the symptom progression of pressure-overload, improves cardiac function, and protects hearts against pressure-induced HF.

Encapsulated Cell Technology-Based Delivery of a Complement Inhibitor Reduces Choroidal Neovascularization in a Mouse Model.

PURPOSE: Age-related macular degeneration (AMD) is a slowly progressing disease, and risk appears to be tied to an overactive complement system. We have previously demonstrated that mouse choroidal neovascularization (CNV) and smoke-induced ocular pathology can be reduced with an alternative pathway (AP) inhibitor fusion protein consisting of a complement receptor-2 fragment linked to the inhibitory domain of factor H (CR2-fH) when delivered systemically. Here we developed an experimental approach with genetically engineered encapsulated ARPE-19 cells to produce CR2-fH intravitreally. METHODS: ARPE-19 cells were generated to stably express CR2 or CR2-fH, microencapsulated using sodium alginate, and injected intravitreally into 2-month-old C57BL/6J mice. CNV was induced using argon laser photocoagulation 4 weeks postinjection. Presence of capsules and progression of CNV was analyzed using optical coherence tomography. Bioavailability of CR2-fH was evaluated in retina sections by immunohistochemistry, and efficacy as an AP inhibitor by C3a ELISA. RESULTS: Secretion of CR2-fH or CR2 from encapsulated ARPE-19 cells was confirmed. An efficacious concentration of CR2-fH capsules to reduce CNV was identified. Bioavailability studies showed that CR2-fH was present in capsules and retinas of injected mice, and reduced CNV-associated ocular C3a production. CONCLUSIONS: These findings indicate that the AP inhibitor CR2-fH, when generated intravitreally, can reduce CNV in mouse. TRANSLATIONAL RELEVANCE: Encapsulated ARPE-19 cells secreting CR2-fH or perhaps other antiangiogenic or prosurvival factors might be useful as a potential therapeutic tool to treat age-related macular degeneration.

Removing vessel constriction on the embryonic heart results in changes in valve gene expression, morphology, and hemodynamics.

BACKGROUND: The formation of healthy heart valves throughout embryonic development is dependent on both genetic and epigenetic factors. Hemodynamic stimuli are important epigenetic regulators of valvulogenesis, but the resultant molecular pathways that control valve development are poorly understood. Here we describe how the heart and valves recover from the removal of a partial constriction (banding) of the OFT/ventricle junction (OVJ) that temporarily alters blood flow velocity through the embryonic chicken heart (HH stage 16/17). Recovery is described in terms of 24- and 48-hr gene expression, morphology, and OVJ hemodynamics. RESULTS: Collectively, these studies show that after 24 hr of recovery, important epithelial-mesenchymal transformation (EMT) genes TGFßRIII and Cadherin 11 (CDH11) transcript levels normalize return to control levels, in contrast to Periostin and TGFß,3 which remain altered. In addition, after 48 hr of recovery, TGFß3 and CDH11 transcript levels remain normalized, whereas TGFßRIII and Periostin are down-regulated. Analyses of OFT cushion volumes in the hearts show significant changes, as does the ratio of cushion to cell volume at 24 hr post band removal (PBR). Morphologically, the hearts show visible alteration following band removal when compared to their control age-matched counterparts. CONCLUSIONS: Although some aspects of the genetic/cellular profiles affected by altered hemodynamics seem to be reversed, not all gene expression and cardiac growth normalize following 48 hr of band removal. Developmental Dynamics 247:531-541, 2018. © 2017 Wiley Periodicals, Inc.