Biodegradable multilayer nanocapsules for the delivery of biologically active agents in target cells

#The instant application contains a Sequence Listing that has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Apr. 18, 2022, is named LNK_207D1_SEQ_LIST.txt and is 672 bytes in size.

#One of the rapidly developing directions of biomedical research and nanotechnology is the design of new delivery systems for bioactive materials and, in particular, of genetic material into target cells. Various synthetic approaches for transfer of coding DNA and regulatory RNA molecules, e.g. short interfering RNA, non-coding RNAs and newly developed CRISP/Cas9 DNA and RNA elements to target cells have meanwhile been demonstrated. Most cancer cell lines can be efficiently manipulated with the help of these systems. However, establishment of the transfer of regulation elements to primary cells and embryonic or adult stem cells is time consuming and frequently accompanied by unwanted side effects hampering translation of in vitro findings to in vivo models and clinical practice.

#For basic research purposes, efficient nontoxic approaches for manipulation of gene expression by knocking down a gene of interest using siRNA and miRNA, or overexpressing a gene of interest by transfer of coding DNA are sought. Whereas the majority of the established immortalized cells lines broadly used by the scientific community allow efficient RNA and DNA transfer using lipid-, polysaccharide-, polymer-, or calcium precipitation-based commercially available reagents, manipulation of gene expression in primary and stem cells remains a challenge. More complexity is expected by the establishment of targeted delivery in animal models and by translation of this technique to clinical practice, since further aspects have to be considered such as a promising drug delivery tool, e.g. regulated duration of substance release, cost-effectiveness and biocompatibility.

#FIGS. 1A and 1B show a characterization of the capsules by scanning electron microscopy (SEM). FIG. 1A shows SEM image of a vaterite CaCO3 particle; FIG. 1B shows an SEM image of core-shell capsules showing spheroid structures of slightly different sizes. The size of capsules is directly dependent of the size of CaCO3 valerites, which is determined by concentration of the reagents, salts solubility, reaction time, and rotation speed during mixing.

#FIGS. 2A and 2B show the preparation of nanocapsules loaded with a fluorescent dye and siRNA. FIG. 2A shows a schematic representation of capsule loading with siRNA: first, two layers of DS and PARG are coated on the CaCO3 core; then siRNA layer can be coated directly on a PARG and covered with an additional PARG layer. As the last step, core can be removed with EDTA; FIG. 2B shows images of capsules loaded with encapsulated Rhodamin B isocyanate (RdnB) and control siRNA, labeled with Alexa Fluor 488 (ctrsiRNA-488); RdnB/(DS/PARG)2/siRNA/PARG). In the red channel, Rhodamin B is visible, showing capsules loaded with RdnB; in the green channel Alexa Fluor 488 can be visualized. Overlay of both images shows a clear merge of both colors giving the color yellow, indicating successful encapsulation of siRNA into the capsules.

#FIGS. 3A and 3B show the efficient uptake and intracellular localization of polyelectrolyte nanocapsules in HT1080 cells. FIG. 3A shows that for determination of optimal capsule concentration 10, 20 and 50 capsules/cell were applied for treatment of HT1080 cells; 24 h post-treatment cells were fixed; stained with phalloidin-Alexa488 and DAPI and subjected to confocal microscopy. Only few capsules could be visualized by application of 10 capsules/cell; concentration of 20 capsules/cell was considered as optimal; whereas 50 capsules/cell exhibited toxic effect, causing nuclei deformation (white arrow heads) and access of not internalized capsules in the solution (white arrows). FIG. 3B shows a comparison of intracellular localization of nanocapsules (left image, showing uptake of capsules loaded with siRNA-Alexa488) and vesicles (right image, showing uptake of vesicles loaded with red dye PKH26). Very similar localization in the perinuclear region suggests that nanocapsules are able to deliver their payloads, e.g. RNA to physiological intracellular sites mimicking extracellular vesicles in their function. Scale bar 50 μm.

#Particularly preferred embodiments of the present invention are shown in the following Examples:

#First, a new protocol allowing production of small biodegradable capsules with efficient encapsulation of high RNA amounts was established. The capsules are shown in FIGS. 1A and 1B. For this purpose, preparation of a CaCO3 core was done using a recently developed protocol (FIG. 1A). Salt concentration and duration of stirring conditions were adjusted to produce particles 100-600 nm diameter (FIG. 1B). Then two biodegradable polymers, dextran sulfate sodium salt (DS) and poly-L-arginine hydrochloride (PARG), were assembled using LbL technique to produce biodegradable capsules.

#The following materials were used: Anhydrous sodium carbonate, sodium chloride, ethylene glycol, calcium chloride, dextran sulfate sodium salt (DS, MW>70 000), poly-L-arginine hydrochloride (PARG, MW>70 000), Rhodamine B isothiocyanate (MW 536.08), phosphate buffered saline (PBS, 0.01M), calcium chloride dihydrate, ethylenediaminetetraacetic acid disodium salt (EDTA), dimethyl sulfoxide (DMSO), were all obtained from Sigma-Aldrich. RPMI-1640 medium, fetal bovine serum (FBS), was purchased from Thermo-Fischer Scientific. Control siRNA labeled with Alexa 488 (ctrsiRNA-488) and AllStar Death Control siRNA (apoptsiRNA) were purchased from Qiagen.