IGF-1/PLGA microparticles – development, characterisation for cardiac applications

Insulin-like growth factor-1 (IGF-1) poly (lactic-co-glycolic acid) (PLGA) microparticles – development, characterization, and in vitro assessment of bioactivity for cardiac applications

Aim: The aim of this study was to evaluate the formulation of a synthetic IGF-1 (pIGF-1) in PLGA microparticles (MP).

Methods: Poly (lactic-co-glycolic acid) (PLGA) MPs loaded with pIGF-1 were prepared, characterised and evaluated using double emulsion solvent evaporation method.

Results: Spherical MPs showed an average particle size of 2 µm, encapsulation efficiency (EE) of 67% and 50% degradation over 15 days. With a view to enhancing retention in the myocardium, the MP formulation was encapsulated in a cross-linked hyaluronic acid hydrogel. pIGF-1 released from MPs and from MPs suspended in hyaluronic acid hydrogel remained bioactive, determined by a significant increase in cellular proliferation of c-kit+ cells.

Conclusion: This formulation has potential for loco-regional delivery to damaged myocardium to promote the survival of cardiomyocytes.

Myocardial infarction (MI) typically occurs following rupture of an atherosclerotic plaque with subsequent thrombus formation, resulting in partial or complete blockage of a coronary vessel. Insufficient blood flow to the heart muscle creates an area of ischaemia, causing mass death of cardiomyocytes. Restoration of blood flow is a key strategy used to reduce scar size, improve long-term myocardial function and reduce patient death. Yet, while reperfusion after MI has significantly reduced mortality, it has led to an increased incidence of chronic heart failure (HF) . Despite advancements in medical therapies to treat HF, survival rates remain poor with >50% mortality within 5 years.

One strategy to reduce negative remodelling post-MI is to target early cardiomyocyte death, decreasing inflammation and loss of viable myocardial tissue.

The pro-survival and anti-apoptotic agent, insulin-like growth factor-1 (IGF-1), has been shown to protect cardiomyocytes from hypoxia-induced apoptosis in vitro through activation of the phosphatidylinositol-3-kinase-AKT (PI3K-AKT) signalling pathway. Administration of one-time low-dose IGF-1 early in the reperfusion stage resulted in potent myocardial salvage, extending to long-term benefits in MI size, wall function and structure in a preclinical model of acute MI.

Recently, the safety of single low-dose intracoronary IGF-1 was established in humans post-MI for the first time. While this clinical trial did not report any improvements in LVEF following treatment with either 1.5 ng IGF-1 or 15 ng IGF-1, it identified a possible dose-dependent benefit on post-MI remodelling that warrants further investigation. Given the short half-life of IGF-1 in circulation, it is conceivable that the cardioprotective effect of exogenous IGF-1 may be optimised by an effective protein carrier system and delivery platform.

Poly (lactic-co-glycolic acid) (PLGA), a copolymer of poly (glycolic acid) (PGA), and poly (lactic acid) (PLA), is a widely studied synthetic biodegradable polymer for the controlled delivery of drugs and macromolecules. However, while the formulation of a sustained release delivery system addresses one challenge, retention of the formulation at the required site of action presents a second question to be addressed. Hydrogels have the potential to increase retention of the MPs in the affected myocardium with previous studies having shown that stem cells encapsulated in hydrogels resulted in manifold increased retention in the heart. Hyaluronic acid (HA) is a naturally occurring linear unbranched glycosaminoglycan polymer and is a common constituent of the extracellular matrix (ECM) in humans. Its inherent biocompatibility and biodegradability characteristics make it favorable for use in tissue engineering, but native HA hydrogels exhibit poor mechanical properties. Cross-linkable derivatives of HA form hydrogels which exhibit an extended biological half-life and withstand much higher mechanical loads making hydrogels formed from such derivatives more suitable for clinical applications.

Here in they describe the development of a formulation which has the potential to offer a mechanism for the delivery, retention and sustained release of pIGF-1in cardiac applications. PLGA microparticles (MPs) using the water in oil in water (W/O/W) double emulsion solvent evaporation method were fabricated. These MPs contained a synthetic IGF-1 protein molecule (pIGF-1) and Human Serum Albumin (HSA) as a stabilising protein as opposed to the more commonly used combination of recombinant IGF-1 (rhIGF-1) and Bovine Serum Albumin (BSA) .

A chemically synthesised protein provides for a more efficient approach to synthesis and purification, leading to higher yields and reduced costs relative to recombinant approaches, and HSA potentially reduces immunogenicity issues, providing for greater clinical translatability. These pIGF-1 MPs were then loaded into hyaluronic acid-tyramide (HA-TA) hydrogels (MPHA-TA) from which the bioactivity of pIGF-1 was assessed after release.1

Schematic representation of work performed in this study. (W/O/W: water in oil in water); pIGF-1: synthetic insulin-like growth factor-1; PLGA: Poly (lactic-co-glycolic acid); HA-TA: Hyaluronic acid-tyramine).

Schematic representation of work performed in this study. (W/O/W: water in oil in water); pIGF-1: synthetic insulin-like growth factor-1; PLGA: Poly (lactic-co-glycolic acid); HA-TA: Hyaluronic acid-tyramine).

Schematic diagram illustrating the preparation of blank (bMPs) and pIGF-1 loaded (pMPs) PLGA microparticles using W/O/W double emulsion solvent evaporation method. Primary emulsion (W/O) was formed by dispersion of dH2O + HAS or pIGF-1 +dH2O + HAS in the organic phase (PLGA in DCM) under sonication for bMPs and pMPs, respectively. A 1%w/v PVA solution was prepared in 1.13%w/v NaCl (PVA aqueous phase). The primary emulsion was added to the PVA aqueous phase (1%w/v PVA in 1.13%w/v NaCl) again under sonication forming a secondary emulsion W/O/W) followed by removal of the solvent by evaporation. MPs were isolated by centrifugation, washed three times with 1.13% w/v NaCl solution, and freeze-dried.

Schematic diagram showing the preparation of a 2% w/v blank (HA-TA) and pMPs loaded (MPHA-TA) hydrogel: lyophilised HA-TA was rehydrated in PBS. Syringes containing Part A (HA-TA-HRP) and Part B (HA-TA-H2O2) were connected to a mixing reservoir and the contents were forced into contact at the mixing reservoir. Hydrogels were collected in the custom-built PTFE moulds. The final concentration of crosslinking agents was 0.12 units/mL HRP and 0.0015%/mL H2O2 (30% v/v). pMPs were suspended in the portion containing the HRP portion of the gel to form MPHA-TA. Pictured also is the formed cross-linked hydrogel.

  1. Aamir Hameed, Laura B. Gallagher, Eimear Dolan, Janice O’Sullivan, Eduardo Ruiz-Hernandez, Garry P. Duffy & Helena Kelly (2019) Insulin-like growth factor-1 (IGF-1) poly (lactic-co-glycolic acid) (PLGA) microparticles – development, characterisation, and in vitro assessment of bioactivity for cardiac applications, Journal of Microencapsulation, 36:3, 267-277, DOI: 10.1080/02652048.2019.1622605