STUDIES ON THE CELLULAR PENETRANCE CAPABILITIES OF THE MEDICINE-LOADED NANOPARTICLES

STUDIES ON THE QUANTIFICATION OF RELEASED INTERNALIZED MEDICINE

       The tests on the internalization of nanoparticles were performed using 3D spheroids and tumoroids of human and murine melanoma and primarily envisaged studying the penetrance capability of nanoparticles along with the quantification of internalized doxorubicin 

The acquired resistance to chemotherapeutic and targeted drugs is an undesirable effect and the most common cause of treatment failure in cancer patients. Malignant melanoma cells have been lately reported as increasingly resistant against therapeutic agents during chemotherapy, leading to the design and development of targeted administration systems that are able to elude drugs resistance, have increased efficacy and exhibit mild secondary adverse effects.

In order to perform the internalization tests on the doxorubicin (DOX)-loaded nanoparticles, we synthesized 3D melanoma spheroids and tumoroids. These were subsequently treated with nanoparticle formulations developed within the framework of this project, the ultimate goal being to reveal and quantify the penetrance capability of DOX-loaded nanoparticles in conditions closely emulating the in vivo testing.

 What the image displays

Prior to the internalization tests, we performed an investigation using the FlowSight imaging cytometer with the purpose of studying the uptake of SPION-citrate-DOX in B16F10 melanoma cells under standard treatment conditions of 2D cultured cells.

This investigation revealed an uptake of DOX into murine melanoma cells, showing enhanced apoptosis. The cells treated with SPION-citrate suffered no significant alterations while those subjected to either DOX or SPION-citrate-DOX showed membrane integrity changes visible under suitable illumination.

The internalization of DOX is visible on channel Ch04 while apoptosis and necrosis can be seen on channels Ch02 (Annexin V-FITC) and Ch05 (PI), respectively. The morphological aspect of cells can be observed on channel Ch01 BF.

STUDIES ON THE CELLULAR PENETRANCE CAPABILITIES OF THE MEDICINE-LOADED NANOPARTICLES

STUDIES ON THE QUANTIFICATION OF RELEASED INTERNALIZED MEDICINE

 The internalization tests were performed using various types of nanoparticles on 3D homotypic and heterotypic spheroids of murine melanoma, envisaging the rigorous determination of the penetrance capacity of each type of nanoparticles along with a quantification of the internalized doxorubicin

B16F1, B16F10 and B16F1 PM Primary Melanoma model cellular lines were used as homotypic spheroids. Prior to any studies, these were marked- at 37°C- with DiO, a highly fluorescent lipophilic dye that gives an even labelling of the cellular membrane, leading to high quality observations under a fluorescence microscope. The heterotypic spheroids entailed CCD dermal human fibroblasts introduced in co-culture.The uptake of SPION-citrate-DOX (68μg/ml) nanoparticles, respectively of the PLGA-PVA-SPION-DOX (2 and 1.7μg/ml) was monitored after 48h of treatment with the aid of the TissueFAXSplus (TissueGnostics) imaging system. The negative control is given by physiological serum while for the positive one free DOX was used (in terms of the highest concentration of DOX bounded to nanoparticles).

  What the image displays

Fluorescence images of B16F1 Primary Melanoma, B16F1 and B16F10 homotypic spheroids, marked with DiO and treated for 48h with nanoparticles pertaining to the SPION-citrate-DOX and to PLGA-PVA-SPION-DOX. The SPION-citrate systems are labelled with ‘SpC’ while those of SPION-citrate-DOX bear the name ‘SpC-DOX’. PP-1e/1f-DOX is the denomination for PLGA-PVA-SPION-DOX, corresponding to concentrations  of 2, respectively 1.7μg/mL. The images were acquired with the aid of a fluorescence microscope, deploying the TissueFAXSplus imaging system, equipped with a PCO Pixelfly digital camera, in 20x magnification. The sample size was 100μm.

These images reveal the internalization of SPION-citrate-DOX nanoparticles into the in vitro 3D homotypic spheroids, confirming the intracellular delivery for the B16F1 PM and the B16F10 lines. The recorded signal for the SPION-citrate-DOX is similar to that pertaining to the positive control. DOX-functionalized PLGA-PVA-SPION nanoparticles yielded no signal and thus  no significant amount of released medicine, regardless of the DOX concentration.  

STUDIES ON THE CELLULAR PENETRANCE CAPABILITIES OF THE MEDICINE-LOADED NANOPARTICLES

STUDIES ON THE QUANTIFICATION OF RELEASED INTERNALIZED MEDICINE

 Following the internalization tests, the quantity of absorbed doxorubicin was determined, assessing the effect of nanoparticles on the proliferation of the murine melanoma cells at the same time.

The internalization studies performed on cellular lysates after 48h of treatment show that lyophilization favors the uptake efficiency. For a proper quantification of the SPION-cit-DOX internalization into the B16F10 murine melanoma homotypic spheroids, as well as for a more accurate determination of the actual amount of DOX uptaken by the cells, the cellular lysates were spectrofluorimetrically measured.

 What the image displays

The upper graph depicts the quantitative internalization of SPION-citrate-nanoparticles into B16F10 cells.

Various comparisons were performed between formulations of bulk SPION-citrate-DOX nanoparticles and those filtered through a 0.22 microns filter (SPION-cit-f-DOX). The results demonstrate that cancerous cells treated with SPION-citrate-DOX exhibit a decrease in proliferation as compared to those treated solely with SPION-citrate, the latter having no effect whatsoever on the cells, even in similar concentrations.

Moreover, the filtered variants induce milder effects. However, even the medicine-free nanoparticles exhibit a negative impact on the cells, most likely due to their reduced dimensions, dimensions that allow for their uptake through other pathways of endocytosis.