Application of drug delivery systems for the controlled delivery of growth factors to treat nervous system injury

ABSTRACT

Nervous system injury represent the most common injury and was unique clinical challenge. Using of growth factors (GFs) for the treatment of nervous system injury showed effectiveness in halting its process. However, simple application of GFs could not achieve high efficacy because of its rapid diffusion into body fluids and lost from the lesion site. The drug delivery systems (DDSs) construction used to deliver GFs were investigated so that they could surmount its rapid diffusion and retain at the injury site. This study summarizes commonly used DDSs for sustained release of GFs that provide neuroprotection or restoration effects for nervous system injury.

KEYWORDS:

• drug delivery systems
• growth factors
• nervous system injury
• neurogenesis

Introduction

The prevalence of people presented with nervous system injury has dramatically increased and thus bring big burden for the society. Medical treatment of nervous system injury, including peripheral nerve injury (PNI) and spinal cord injury (SCI), as well as brain injury, currently pose a significant challenge worldwide.¹ The regenerative ability of the nervous system after injury was often limited.² In the past decades, great effort has been made for the treatment of nervous system injury.^3,4

Application of growth factors (GFs) is a promising approach to nervous system injury. GFs are biologically active polypeptides that bind to their receptors and activate a cascade of molecular events.⁵ The major GFs used for nervous system injury are nerve growth factor (NGF), platelet derived growth factor (PDGF), brain derived neurotrophic factor (BDNF), basic fibroblasts growth factor (bFGF), ciliary neurotrophic factor (CNTF), glial cell-derived neurotrophic factor (GDNF), transforming growth factor-β (TGF-β), neurotrophin-3 (NT-3) and vascular endothelial growth factor (VEGF) families.

Although GFs play important roles in nervous system injury regeneration process, simple application of GFs present some critical limitations of its efficiency, because GFs can diffuse rapidly from the injury site into the body fluids.⁶ Besides that, the half-life of GFs is short due to their instability in vivo. GFs, physiologically produced or locally injected, can be easily degraded by proteases which are activated in the body fluids. Thus, usually used methods are large doses or repeated injections to achieve sustained clinical effect. But high doses of GFs may result in serious side effects such as thrombocytopenia, renal toxicity or even the development of certain malignant cells.⁷ Modified viruses can be vectors that carry GFs genes to the injury site of nervous system in multiple injury models. The introduction of GFs into the brain, spinal cord and peripheral nerve by viral vectors including lentivirus, herpes simplex virus type-1 (HSV-1) and AAV is being explored.^8,9 Shortcoming of the approach is the need to inject the vectors into the central nervous system that may give rise to immunological rejection and also leads to safety concerns. Administration of GFs using an osmotic mini-pump system, intrathecal catheters or other injection devices may lead to chronic nerve compression or fibrotic capsule formation and thus requires a second operation to remove devices, a second operation increases the risk of infection.¹⁰ Physical adsorption of GFs on biomaterials or simple encapsulation of GFs into biomaterials resulted in complete GFs release either in an initial burst or within a few days.

To overcome these concerns, different kinds of drug delivery systems (DDSs) are being pursued to surmount the diffusion of GFs in the flushing of body fluids or improve the stability of the GFs, thus improve the treatment effectiveness of GFs.^11,12 DDSs retain sufficient concentrations of GFs at the lesion sites for a long time and control the spatial and temporal delivery of GFs.¹³ The retained GFs enable migration of cells to sites of injury following proliferation and differentiation during the regeneration process.¹⁴ The DDSs largely limit the release of GFs to non-target tissue, so this method can decrease side effects.¹⁵

Biomaterials are one of the most important elements which construct DDSs.^16,17 The successful use of biomaterials to be carriers for GFs has inspired a great deal of interest in their use for tissue engineering strategies.^18–20 In the selection of biomaterials for delivering of GFs, the nature of the implantable biomaterials possess should be taken into consideration. The biomaterial should be biodegradable carrier material since non-degradable material may lead to chronic nerve compression or fibrotic capsule formation around the lesion site.²¹ Biomaterials should also possess the characteristics of non-cytotoxic, non-immunogenic and high loading efficiency of GFs.²² After loading of GFs on biomaterials, the biological activity of GFs should not be affected.²³

Several clinically available biomaterials have been approved by Conformit Europe (CE) and U.S. Food and Drug Administration (FDA) including: polyglycolic acid (PGA), poly-DL-lactide-caprolactone (PLCL), collagen type I.²⁴ Among biomaterials adopted in an attempt to enhance neural regeneration and functional recovery, collagen was widely used because of its good biocompatibility and excellent biodegradability.^25–27 Collagen is one of the extracellular matrix (ECM) molecules, has the special advantage of possessing cell-adhesive or signaling domains, which made collagen material provide a more suitable regeneration microenvironment.²⁸ Because of its evolutionary conservatism is high in vertebrates, the antigenicity of collagen is very low. Therefore, allogeneic collagen implants produce very low immunity in vivo even in the long term. Besides, the functional groups of collagen can be easily modified, which is the basis for controlling the rate of degradation by cross-linking in vivo.

The methods of immobilizing GFs on collagen materials have been reported in recent years. In our previous study “Sustained delivery of glial cell-derived neurotrophic factors in collagen conduits for facial nerve regeneration” in the journal Acta biomaterialia, we immobilized GDNF in collagen conduits as GDNF delivery system and delivered bioactive GDNF in a localized and sustained manner for facial nerve regeneration.²⁹ In the study, we first prepared interconnected porous collagen conduits and then immobilized GDNF on the collagen surface by chemical conjugation. Collagen contains large amount of hydroxyproline, amines, and glycine, which is rich in primary amines(-NH₂).³⁰ Traut’s Reagent (2-Iminothiolane) reacts with primary amines of collagen material to introduce sulfhydryl (-SH) groups, which prepared it for conjugating with sulfo-SMCC. Sulfo-SMCC is a heterobifunctional crosslinker accompanied with Nhydroxysuccinimide (NHS) ester and maleimide groups. The NHS esters of sulfo-SMCC allow covalent conjugation of –NH₂- while maleimide groups allow covalent conjugation of -SH-containing molecules. Based on the properties of sulfo-SMCC, –NH₂ of GDNF reacted with the NHS esters of sulfo-SMCC to form amide bonds and then -SH groups on the collagen reacted with maleimides of sulfo-SMCC to form stable thioether bonds. After this conjugation process, the results of ELISA showed that the GDNF delivery system largely reduced dispersion of GDNF from the immobilization site. Furthermore, conjugation with Traut’s reagent and sulfo-SMCC did not reduce the biological activity of GDNF. After transplantation of the GDNF delivery system in vivo for the treatment of facial neve transection injury, the degree of nerve regeneration was close to that of the autograft group.

In another study, collagen binding domain (CBD) was fused to the N-terminal of BDNF to prolong the presence of BDNF on collagen materials.³¹ CBD is a polypeptide “TKKTLRT” that have special binding ability to collagen.³² The gene encoding the fusion proteins “CBD-BDNF” was amplified by PCR to construct expression vector which was then transformed into Escherichia coli BL21 (DE3) strain. The expression of fusion proteins was induced by isopropyl β-D-thiogalactopyranoside (IPTG). Through this method, the amino acid sequence of CBD was fused to the N-terminal of the amino acid sequence of BDNF and formed the fusion proteins. The CBD-BDNF exhibited comparable bioactivity in contrast to native BDNF and maintained the specific laminin binding ability on the linear ordered collagen scaffolds (LOCS). ELISA assay shown that CBD could help the controlled release of the CBD fusion protein from collagen scaffolds effectively. The DDSs consisting of LOCS loaded with CBD-BDNF promoted better neural regeneration as well as functional recovery. Similarly, the genetically engineered EGF that fused with CBD maintained EGF at a sufficiently-high level on the surface of collagen hydrogel for a long period. The prolonged presentation of EGF was effective for promoting the proliferation of neural stem cells (NSCs) in collagen hydrogel.³³

The GFs could also be immobilized on collagen material after its activation by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) reaction solution. In the presence of EDC, N-hydroxysulfosuccinimide (Sulfo-NHS) could react with carboxyl groups (–COOH) of collagen and form semi-stable amine-reactive NHS ester. After that, the semi-stable Sulfo-NHS ester can react with –NH₂ of GFs to form stable amide bond. This method retains an effective amount of GFs at the injury site to enhance the regeneration of the tissues. In a previous study³⁴, VEGF and angiopoietin-1 (Ang1) were covalently immobilized on porous collagen scaffolds via EDC to promote rapid vascularization of engineered tissues. Results showed that the immobilized GFs enhanced rat aortic endothelial cells proliferation and tube formation. After nervous system injury, the destruction of blood vessel integrity at the lesion site resulted in decreased blood and oxygen supply following poor axonal regrowth, cell death and cavity formation.³⁵ Thus, improved angiogenesis at the lesion site after nervous system injury was beneficial for neural regeneration.

Some GFs have high affinity to heparin, so collagen can be modified by heparin to create the binding ability of GFs to collagen materials.^36,37 In another study, heparin was crosslinked to collagen sponges by EDC and NHS, and subsequently bFGF was adsorbed to the heparin modified collagen sponges.³⁸ Human umbilical vein endothelial cells (HUVECs) were seeded on collagen sponges to evaluate bioactive maintenance of bFGF after modification with heparin. Meanwhile, the sustained release profile was tested at different time point using an ELISA assay. Results in their study suggested that the heparinized collagen sponges exhibited relatively small initial burst release and the release of bFGF from the DDSs was sustained over 37 days. Besides that, HUVECs proliferated and migrated well and distributed uniformly on heparinized collagen sponges. The heparinized collagen sponges group exhibited the least loss of bFGF activity in contrast to other groups.

Discussion

Although the above methods of constructing DDSs by collagen immobilized with GFs had achieve great progress in nervous system injury repairing, the regeneration extent was still inferior to the positive group (autograft group or sham group) in resent studies. It is because DDSs can’t provide glial cells, physical guiding cues, and GFs to the same level as positive group treatment during the regeneration process. Thus, application of multiple GFs and neural cells represent a step further method for the neural regeneration of engineered neural tissues.

Due to collagen possesses the characteristics of low antigenecity, excellent biocompatibility and biodegradability, collagen material has been studied in clinical trials to determine its safety and effectiveness. Xenogeneic collagen matrix from porcine was used for the generation of keratinized tissue around teeth in a single-masked, randomized, controlled, split-mouth study of 30 patients.³⁹ The results showed that the porcine collagen matrix increased keratinized tissue around teeth to a similar degree in contrast to the positive control, an autogenous free gingival graft. The study indicated the porcine collagen matrix appeared to be a suitable substitute for autogenous free gingival graft for the patients. However, the application of collagen derived DDSs for nervous system injury repairing in patients remains to be researched in the future work.

Disclosure Statement

No potential conflicts of interest were disclosed.

Additional information

Funding

This work was supported by grants from MST (SQ2018YFA010113) and NSFC31771491, and grants (81471242, 81601069) from the National Nature Science Foundation and Ministry of Science and Technology of China.