(68e) Temporally Controlled Release of Platelet-Rich Plasma from Peg Microgels with Tunable Biodegradation Rate and Size

Biomedical applications such as drug delivery and tissue engineering often require polymeric hydrogels which degrade predictably under physiological conditions. Biodegradable injectable drug delivery systems present attractive avenue for site specific and minimally invasive administration, coupled with precise temporal control over drug release, which can be employed for multiple applications. Specifically, poly (ethylene glycol) (PEG) has been thoroughly investigated for the design of biodegradable hydrogel networks because of its exceptional biocompatibility and inertness. In this study we designed biodegradable PEG microgels with a wide range of degradation kinetics using Michaelâ??s addition chemistry. We further used these microgels for encapsulating and controlling delivery of proteins derived from platelet rich plasma (PRP). PRP is a concentrated assortment of growth factors and cytokines derived from the patientâ??s own platelets. PRP has been proven useful in a number of regenerative medicine applications. However, an effective method to achieve sustained delivery of PRP is still required.

For the design of degradable PEG hydrogels we used multiarm PEG-acrylate (PEGAc) of varying molecular weight and number of arms (4-arm of 10 kDa, to 8-arm of 20 kDa) and three categories of dithiol crosslinkers, namely, ester containing, non-ester containing and dithiols with neighbouring functional groups of different electronegativities. The PEG hydrogel physical and mechanical characteristics could be modulated depending upon the reaction parameters and gelation time for multiarm PEGAc and dithiol crosslinker structure and molecular weight. By changing these parameters, hydrogels with controlled degradation ranging from 10 hours to 32 days and having mesh size in the range of 9 to 14 nm were obtained. We identified two sets of conditions which yielded fast gelling-fast degrading hydrogels and slow gelling-slow degrading hydrogels. Uniquely, the hydrogel storage moduli could be controlled by the dithiol crosslinker chemical identity independent of the degradation time or mesh size of the hydrogel.

Further, we used the optimized combinations of PEG hydrogels to make PEG microgels of sizes varying between 50 to 700 µm by manipulating several key electrospraying parameters. We also investigated various techniques and times suitable for storing protein-loaded PEG microgels. The microgels were stored at seven conditions: 25°C, 4°C, -80°C, Lyophilization (Lyo), -80°C/Lyo, -80°C/DMSO, and -80°C/DMSO/Lyo for 1, 4, 7, and 28 days. DMSO was introduced at 10% (v/v) in 1x PBS. Our results indicated that neither storage modulus nor the structure of incorporated model protein was affected post storage at different conditions. However, comparing the percent distribution of microgel size pre- and post-storage, we found an increase in the mean diameter indicating increased swelling post-storage. Furthermore, environmental scanning electron microscopy (ESEM) images showed that large pores were created by some of the conditions, mainly by lyophilization.

Finally, we tested the PEG hydrogels and microgels for encapsulation and delivery of PRP-derived growth factors. PRP at 2-10% w/v was encapsulated during gelation of the hydrogels or microgels. Release of growth factors was quantified by multiplexer analysis. Biological activity of PRP released from the hydrogels or microgels were tested in vitro using human dermal fibroblast (HDF) or by circular dichroism, respectively. Direct encapsulation of model proteins in the PEG hydrogels during hydrogel gelation preserved their secondary structure. Release kinetics for several model proteins was found to be dependent on the protein size, as well as mesh size and degradation rate of the hydrogel. The amount of total protein release increased over time, corresponding to increase in hydrogel mesh size. Multiplexer growth factor analysis of the releasates showed continuous release of growth factors (PDGF-BB, EGF, RANTES) from PRP-containing hydrogels until degradation. PRP releasates also demonstrated increased proliferation in HDF cells over a period of 72 hours, comparable to that of a bolus PRP injection. Furthermore, similar results could be translated to PEG microgels obtained by electrospraying.

Thus it can be concluded that we could obtain a range of biodegradable PEG hydrogels by a combination of number of PEG arms and dithiol crosslinkers and reaction pH. Further, these hydrogels could be made into injectable microgels which allowed localized and controlled delivery of PRP growth factors in the active form.