CHARACTERIZATION OF CELL-MIMETIC DRUG CARRIERS FOR SUSTAINED DELIVERY OF THERAPEUTICS

Abstract

Metastatic cancer is a challenging disease to treat. One contributing factor to consider is the selective permeability of the lymph nodes, which enables them to act as a safe haven for cancer cells in the body. One approach is to utilize a Trojan horse strategy, where cryo-shocked T-lymphocytes (CSTLs) act as an engineered cell-mimetic drug carrier to home to the lymph node. CSTLs loaded with small-molecule drugs resulted in rapid release kinetics, which limited achievable doses at the target sites.

To follow on, it was hypothesized that by incorporating a hydrogelated core into the CSTLs system, modifying the CSTLs would provide controlled, delayed, sustained release of the therapeutic agents. This version of the carriers is called hydrogelated T-lymphocytes (HTCs), utilizing DOX-laden poly(ethylene glycol)-based gelation solution (PEG-diacrylate (PEG-DA), initiated with 2-hydroxy-4'-(2-hydroxyethoxy)-2 methylpropiophenone).

Three aims were researched for the hypothesis: (1) characterization of loading efficiency and max loading of CSTLs and HTCs, (2) determination of the release kinetics of CSTLs and HTCs loaded with Doxorubicin, and (3) test target efficiency of loaded drugs against cancer cells in vitro.

To characterize loading efficiency and max loading of CSTLs and HTCs, the ability for HTCs to be loaded with doxorubicin had to be assessed, both visually with an epifluorescence microscope and utilizing a plate reader. The CSTLs have an average loading efficiency of 74.27 ± 6.61%. 10% and 20% PEG-DA in HTCs had average loading efficiencies of 3.95 ± 3.42% and 28.76 ± 10.52%. When testing for maximum loading, respectively for each concentration 0.525 mg/ml, 0.7 mg/ml, and 0.8 mg/ml, the efficiencies were 3.95 ± 3.42%, 12.93 ± 3.42%, and 17.19 ± 2.25%. This aim helped to better understand the loading efficiencies of HTCs and how to increase their loading abilities.

To investigate the release kinetics of CTSLs and HTCs, release experiments of all drug delivery vehicles were completed. The release profiles were analyzed utilizing one and two-phase decay, and the rates of release were determined, after varying controlled release compared to CSTL’s rapid release. Both 20% and 10% PEG-DA HTCs drug delivery vehicles have similar trends, exhibiting one-phase decay graphs. 10% PEG-DA HTCs have a rate of release (K=1.16*102), whereas 20% PEG-DA HTCs have a lower rate (K=7.87*10-5). The 20% PEG-DA HTCs have a higher percentage released than the 10% PEG-DA HTCs. With a lower loading efficiency, the 20% PEG-DA has less to release than the 10% PEG-DA HTCs.

To test the target efficiency of the HTCs loaded with the drug, the loaded HTCs were tested on 4T1s cancer cells. An assay was run to determine the viability of the cancer cells to see how efficiently the drug-loaded vehicles treated them. The viability of the 4T1s on the bottom of the well was analyzed after 20% PEG-DA HTCs released DOX from a transwell directly on the 4T1s. The viability of the 4t1's was 90.7 % after 7 days, compared to the positive and negative controls, which were respectively 100% and 58.5%.

This study demonstrated that the encapsulation of small-molecule drugs inside a hydrogel core (HTCs) produced a drug delivery vehicle with tunable properties that will enable a delayed and controlled release of small-molecule payload. This study supports the hypothesis that incorporating a hydrogelated core into the CSTLs system would provide controlled, delayed, sustained release of the therapeutic agents. This new delivery system potentially creates formulations that enable sustained release of therapeutics locally within the lymph node. Optimizing the amount of drug at the site of the disease, decreasing dosing frequency, and less off-target accumulation leads to lower systemic toxicity for the patient.