Abstract
BackgroundHuman platelets (PLT) and PLT-extracellular vesicles (PEV) released upon thrombin activation express receptors that interact with tumour cells and, thus, can serve as a delivery platform of anti-cancer agents. Drug-loaded nanoparticles coated with PLT membranes were demonstrated to have improved targeting efficiency to tumours, but remain impractical for clinical translation. PLT and PEV targeted drug delivery vehicles should facilitate clinical developments if clinical-grade procedures can be developed.MethodsPLT from therapeutic-grade PLT concentrate (PC; N > 50) were loaded with doxorubicin (DOX) and stored at − 80 °C (DOX-loaded PLT) with 6% dimethyl sulfoxide (cryopreserved DOX-loaded PLT). Surface markers and function of cryopreserved DOX-loaded PLT was confirmed by Western blot and thromboelastography, respectively. The morphology of fresh and cryopreserved naïve and DOX-loaded PLT was observed by scanning electron microscopy. The content of tissue factor-expressing cancer-derived extracellular vesicles (TF-EV) present in conditioned medium (CM) of breast cancer cells cultures was measured. The drug release by fresh and cryopreserved DOX-loaded PLT triggered by various pH and CM was determined by high performance liquid chromatography. The thrombin activated PEV was analyzed by nanoparticle tracking analysis. The cellular uptake of DOX from PLT was observed by deconvolution microscopy. The cytotoxicities of DOX-loaded PLT, cryopreserved DOX-loaded PLT, DOX and liposomal DOX on breast, lung and colon cancer cells were analyzed by CCK-8 assay.Results15~36 × 106 molecules of DOX could be loaded in each PLT within 3 to 9 days after collection. The characterization and bioreactivity of cryopreserved DOX-loaded PLT were preserved, as evidenced by (a) microscopic observations, (b) preservation of important PLT membrane markers CD41, CD61, protease activated receptor-1, (c) functional activity, (d) reactivity to TF-EV, and (e) efficient generation of PEV upon thrombin activation. The transfer of DOX from cryopreserved PLT to cancer cells was achieved within 90 min, and stimulated by TF-EV and low pH. The cryopreserved DOX-loaded PLT formulation was 7~23-times more toxic to three cancer cells than liposomal DOX.ConclusionsCryopreserved DOX-loaded PLT can be prepared under clinically compliant conditions preserving the membrane functionality for anti-cancer therapy. These findings open perspectives for translational applications of PLT-based drug delivery systems.
Highlights
Human platelets (PLT) and PLT-extracellular vesicles (PEV) released upon thrombin activation express receptors that interact with tumour cells and, can serve as a delivery platform of anti-cancer agents
Cryopreserved DOX-loaded PLT can be prepared under clinically compliant conditions preserving the membrane functionality for anti-cancer therapy
The acidic pH, the presence of cancer cells and tissue factor-expressing cancer-derived extracellular vesicles (TF-EV), and the thrombin-rich milieu, which is typical of all tumours, contribute to PLT activation, PEV generation, and release of DOX, in a complementary cancer cell killing loop
Summary
Human platelets (PLT) and PLT-extracellular vesicles (PEV) released upon thrombin activation express receptors that interact with tumour cells and, can serve as a delivery platform of anti-cancer agents. The “vicious” pathogenic attraction existing between tumour cells and PLT membranes has recently stimulated the engineering of targeted PLT membrane-inspired nanocarriers aiming at delivering anti-cancer agents, following a so-called “Trojan Horse” strategy In this approach, the PLT membrane machinery serves as a camouflage against the phagocytic system and is used to design smart drug delivery systems (DDS) with enhanced targeting ability to tumour cells [19,20,21,22,23,24]. Drugloaded nanoparticle/nanogel preparations coated with whole PLT membranes exerted potent toxicity against tumour cells and lowered the risk of metastasis in animal models [20, 28, 29] Those studies are crucial proof-ofconcept and learning tools in understanding the capacity of the bioreactive PLT membrane machinery to target cancer cells. Our study unveils the unique and superior ability of cryopreserved intact PLT to serve as a “Trojan Horse” bioreactive delivery carrier of cancer drugs, through generation of potent PEV and enhanced release at low pH and in the presence of cancer cell-derived TF-EVs
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