Purpose: In occlusive vascular diseases, thrombus dissolution and rapid restoration of blood flow is critical in preventing severe consequences like heart attacks or stroke. Currently, this reperfusion is clinically achieved by intravenous administration of fibrinolytic agents like tissue plasminogen activator (tPA). Though effective, the systemic delivery of tPA suffers from several pharmacokinetic limitations and hemorrhagic side-effects. As a result, there is a pressing need for the development of therapeutic platforms that can target certain clot-specific cells or biomolecules and deliver the potent thrombolytic drug selectively at the desired site. Platelets provide an excellent paradigm for such design owing to their effective margination behavior, firm and specific adhesion to clot, and clot modulation ability. Inspired by these cues from natural platelets, we have developed tPA encapsulated platelet-mimetic carriers. We hypothesize that these soft, discoidal targeted delivery vehicles can efficiently interact with the activated platelets at the clot site and release tPA locally at a controlled rate.
Methods: To develop tPA carriers with platelet-like morphology, layer-by-layer (LbL) deposition technique was used with tPA-loaded calcium carbonate microsphere templates. Briefly, in situ encapsulation of tPA was achieved during the synthesis of calcium carbonate microparticles using calcium choride and sodium carbonate solutions. This was followed by alternate electrostatic deposition of anionic bovine serum albumin (BSA) and cationic poly allyl amine hydrochloride (PAH), along with covalent crosslinking of the layers with glutaraldehyde. The coated spheres were then incubated in 0.2M ethylenediaminetetraacetic acid (EDTA) for 1 hour to dissolve the calcium carbonate cores and yield soft, hollow, discoidal microcapsules for drug encapsulation. Loading and stability studies were performed on the tPA-loaded microcapsules using ELISA. Furthermore, the microcapsules were conjugated to fluorescent KRGDWC peptide via PAMAM dendrimers for targeting the activated platelets in the clot.
Results: tPA loading was quantified by performing total tPA antigen ELISA on the supernatant from the reaction mixture post-calcium carbonate particle formation. The encapsulation efficiency was found to be 66.2 ± 8%, which is in agreement with that of previously reported tPA carriers (Fig 1). Furthermore, tPA activity ELISA was performed on the solution obtained by dissolving uncoated template particles in EDTA. It was found that 78.4±5% activity of the encapsulated tPA was retained during the encapsulation and core dissolution process. To ensure complete coating of the calcium carbonate cores, zeta potential measurements were made after each layer deposition. Reversal of zeta potential indicates complete coating with the polyelectrolyte. In order to visualize the encapsulated drug, similar synthesis of microcapsules was performed with Texas red dextran, a fluorescent model compound with similar molecular weight and charge as tPA. Confocal microscopy was used to image the dextran encapsulated in the core of FITC-BSA/ PAH shell of the microcapsules. Atomic force microscopy measurements showed that the elastic modulus of these soft microcapsules is 23.8 ± 1.84 kPa, which is similar to that of natural activated platelets.
Similarly, for the visualization and quantification of the conjugated peptide on the capsule surface, the thiol group of the cysteine amino acid was conjugated with FITC maleimide and the product was purified using C18 reverse phase columns, followed by characterization with Matrix Assisted Laser Desorption/ Ionization (MALDI). FITC-KRGDWC was then conjugated with succinamic acid PAMAM dendrimers using CDI chemistry and quantified using fluorescence and MicroBCA assay, respectively and 86.5±9.3% by mole peptide was obtained in the purified product. Further, these conjugates were attached to the particle surface using EDC-NHS chemistry and its efficiency was quantified using peptide fluorescence, since BSA shell interferes with the MicroBCA assay. It was found that (20.0 ± 0.4) x 1017 peptide molecules were attached per mg of the microcapsules.
Conclusion: Thus, we have successfully developed soft, discoidal targeted tPA carriers that effectively mimic the physico-mechanical features of natural platelets, to facilitate enhanced margination towards the vascular wall, can potentially target and anchor to the occlusive clot site through interaction with activated platelets in the clot and can bring about controlled tPA release for fibrinolytic action.
Samir Mitragotri– Harvard University