(M1530-07-37) Bioengineered Smart Microcapsules Containing Natural Killer Cells for the Treatment of Pediatric Cancers

Purpose: Natural killer (NK) cells are cytotoxic to tumor cells and are of great therapeutic importance for pediatric and rare cancer therapies¹. They have high activity, lower toxicity (compared to other cancer therapies), and allogenic advantages. In pediatric leukemias, adoptive transfer of NK cells can bridge children not in remission to transplant. The potential to treat pediatric cancers can be enhanced using our bioengineered "smart microcapsules" containing NK cells². This technique allows to increase the local concentration of the NK cells, enhancing their target tumor cell lysis activity. It will also increase the duration of action of the NK cells at the tumor site while temporarily encapsulating them and protecting them from the hostile tumor microenvironment. This study aims to formulate a regenerative medicine-based cell therapy approach to influence the localized microenvironment of hypervascularized tumors by creatively delivering NK cells in bioengineered microcapsules.

Methods: Human natural killer leukemia cells, KHYG1, were cultured in a complete culture medium (Dulbecco's Modified Eagle's Medium F-12 with 20% Fetal Bovine Serum and Interleukin-2). To fabricate the microcapsules, we used a modified Buchi spray dryer B-190 that allows fast manufacturing of microcapsules. KHYG1 cells were suspended in the polymer matrix of aqueous sodium alginate containing trehalose to stabilize the cell membrane. The cell suspension was sprayed using the modified spray dryer through a 0.7 mm nozzle into a calcium chloride solution. The microcapsules were then transferred to chitosan glutamate solution to coat the alginate membrane. The microcapsule suspension was then washed with phosphate buffer saline and centrifuged twice at 190 g (800 RPM) to remove the excess chitosan glutamate. The microcapsules obtained were then transferred to the culture media and stored at 37°C and 5% CO₂ in the incubator. The efficiency of the microencapsulation process was calculated as the percentage of viable cells after the encapsulation process. The microcapsules were characterized for size, shape, and morphology using light and fluorescence microscopy. The cells were stained with a nuclear stain DAPI and observed using a fluorescence microscope to observe cell encapsulation. The parameters affecting the size of the microcapsules, i.e., the compressed air flow rate, pump speed controlling the flow rate of alginate-cell suspension, nozzle diameter, and concentration of sodium alginate solution, were varied to optimize the size of the microcapsules. FTIR-ATR spectra of sodium alginate and the microcapsules were recorded at a resolution of 4 cm^-1 and between 400 to 4000 cm^-1 to observe the formation of cross-linked calcium alginate. The short-term stability of the microcapsules was assessed. Immunoisolation and selective permeability of the microcapsules were confirmed via micro-BCA protein assay.

Results: The efficiency of the microencapsulation process was calculated as a percentage of viable cells after the encapsulation process and was found to be 75%. The microcapsules were nearly spherical, with no deformities. Encapsulation of cells in the microcapsules was confirmed using microscopic images. The size of microcapsules is reduced by increasing the flow rate of compressed air, increasing pump speed controlling the flow rate of alginate-cell suspension, and decreasing the nozzle diameter and concentration of sodium alginate solution. The airflow rate of 350 L/h, pump speed of 9 RPM, nozzle diameter of 0.7 mm, and 0.5 % w/v solution of sodium alginate produced microcapsules of 50-60 µm. The size distribution determined for the optimized batch of microcapsules followed the Gaussian distribution. The FTIR-ATR spectra confirmed the crosslinking of sodium alginate with calcium chloride to form calcium alginate. The cell viability study revealed that KHYG1 cells were viable inside the microcapsules suspended in the media for 10 days. Micro BCA protein assay showed that a large molecular weight protein, i.e., BSA (molecular weight 66.5 kDa), could not cross the microcapsule membrane. This confirmed the selective permeability of the microcapsule membrane.

Conclusion: We could successfully formulate microcapsules encapsulating live KHYG1 cells with narrow size distribution via a modified spraying device. The microcapsules were found to be stable, and encapsulated cells were viable inside the microcapsules for 10 days. The microcapsules were selectively permeable and thus could provide adequate protection to encapsulated cells. The polymer matrix offers the opportunity for a more robust clinical impact as the local concentration of the NK cells can be sustained over a longer duration, thereby minimizing systemic toxicity. Taken together, microcapsules encapsulating KHYG-1 cells thus have the potential to serve as an adjunct treatment approach for pediatric cancers. This envisioned technology aims to bring cellular immunotherapy into Interventional Oncology, wherein localized delivery of small chemotherapeutic molecules is a routine for hyper-vascular tumors.

References: 1. Kimpo, M. S., Oh, B. & Lee, S. The Role of Natural Killer Cells as a Platform for Immunotherapy in Pediatric Cancers. Curr. Oncol. Rep. 21, 93 (2019).
2. Joshi, D. J., Chitre, N. M., Bansal, A., Murnane, K. S. & D’Souza, M. J. Formulation and Characterization of Microcapsules Encapsulating PC12 Cells as a Prospective Treatment Approach for Parkinson’s Disease. AAPS PharmSciTech 22, 149 (2021).

Bioengineered “smart microcapsules” containing natural killer cells for the treatment of pediatric cancers.



Light microscopic image of KHYG-1 cells (40X), and microcapsules encapsulating KHYG-1 cells (10X and 40X)



Microcapsule size distribution (n=25)