Targeted delivery of a PROTAC induced PDEδ degrader by a biomimetic drug delivery system for enhanced cytotoxicity against pancreatic cancer cells

Abstract

Pancreatic carcinoma (PC) has one of the highest mortality-to-incidence ratios of any solid tumor worldwide. Although KRAS mutation is commonly found in 95% of PCs, directly targeting KRAS remains to be a highly challenging task because of its lacking catalytic pockets where molecule inhibitors can bind with. Proteolysis-targeting chimeric (PROTAC) represents an effective approach for specific degradation of disease-causing proteins by hijacking the endogenous ubiquitin-proteasome system (UPS). Previously, we designed a first-in-class PROTAC induced PDEδ degrader (PIPD), which demonstrated improved anti-tumor efficacy against KRAS mutant malignancies. However, translating cellular degradative effects from bench to beside remains a highly challenging task because of PROTAC's poor penetration efficiency across target cytomembranes and non-targeting delivery induced undesired "off target" side-effects. Herein, a smart nano-drug delivery system (CM8988-PIPD) was successfully constructed by biomimetic strategy for targeted delivery of PIPD. The biomimetic nanoparticle showed well-defined regular spherical structure with an average particle size of approximately 124.8 nm. Cancer cytomembrane camouflage endows CM8988-PIPD with excellent in vivo serum stability, controlled drug release profile, favorable biocompatibility & immunocompatibility, and prominent targeting ability to homologous PC cells. Owing to these advantages, the smart DDS significantly enhanced PDEδ degrading efficacy, resulting in induced cellular apoptosis (more than 50% for both PC cells) and suppressed cell proliferation via the inhibition of RAS signaling. In vitro studies illustrated that CM8988-PIPD hold great potential for the treatment of PC, which merits further investigation in both pre-clinical and clinical investigations in the future.

Keywords: KRAS mutation; PDEδ; Pancreatic carcinoma; biomimetic drug delivery system; proteolysis-targeting chimeric.

Figure 1

Schematic illustration of homotypic cell membrane-cloaked biomimetic nanocarrier for targeted delivery of PIPD and its anti-tumor mechanism against PC cells. Enhanced anti-cancer efficacy can be attributed to the synergistic effects of (1) targeted delivery of encapsulated PIPD molecules to tumor tissues by favorable serum stability, biocompatibility and immunocompatibility; (2) elevated tumor uptake of bionic nanosized DDS via homologous cell membrane recognition; (3) inhibition of KRAS signal transduction pathway by efficient PDEδ degradation through hijacking the UPS system by released PIPD molecules in target cells.

Figure 2

Characterizations of CM8988-PIPD DDS. (A, B) Hydrodynamic size of CM8988-PIPD and empty CM8988 vector by DLS. (C) Surface zeta potential of CM8988-PIPD and empty CM8988 vector. (D) TEM morphology of CM8988-PIPD. Scale bar: left 0.5 μm, right 200 nm. (E-G) UV-vis absorption (E), FTIR spectra (F), and XPS spectrum (G) of CM8988-PIPD and counterparts. (H) SDS-PAGE protein analysis for cell lysates, CM8988 empty ventor and CM8988-PIPD DDS. Data are expressed as mean ± SD (n=3).

Figure 3

A. Excellent in vitro serum stability of CM8988-PIPD nanoparticals: DMEM containing 50% (v/v) FBS was employed as an in vitro serum model and the size distribution of CM8988-PIPD was measured by DLS during 6 days. B. Cumulative release of PIPD molecules from CM8988-PIPD DDS at different conditions. Data are expressed as mean ± SD (n=3).

Figure 4

Excellent in vitro biocompatibility and immunocompatibility of biomimetic DDS. A. Hemolysis rate of red blood cells in different concentrations of free and CM8988 encapsulated PIPDs. B. The cell viability of human normal pancreatic ductal epithelial cells (HPDE6-C7) treated with different concentrations of CM8988-PIPD. C. CLSM images of J774A.1 macrophages co-culture with free and CM8988 encapsulated QDs for 4 h. Green and blue fluorescence indicates QD and nucleus, respectively. Scale bar: 20 µm.

Figure 5

Assessment of cellular uptake of biomimetic DDS by FCM and CLSM. A. Effects of cytomembrane on the intracellular uptake of QDs by homologous malignant pancreatic cells (PATU-8988 and PL-45) by FCM. B. Quantitative analysis of FCM results. Mean fluorescence intensity was counted using FlowJo software. Data are mean ± SD (n=3), **P<0.01. C. CLSM images of PATU-8988 and PL-45 cells co-culture with free and cytomembrane encapsulated QDs. Green and blue fluorescence indicates QD and nucleus, respectively. Scale bar: 20 µm.

Figure 6

In vitro cytotoxicity evaluation of biomimetic DDS. (A) Cellular apoptosis of PATU-8988 and PL-45 cells treated with CM8988-PIPD and counterparts by FCM. (B) Quantitative analysis of FCM results. Apoptotic cells were defined as Annexin V⁺/PI^± cells. (C) Bio-TEM images of apoptotic PATU-8988 (up panel) and PL-45 (down panel) cells. The red arrows indicated typical morphologies of apoptosis including chromatic agglutination and formation of apoptotic body. Cell viability of (D) PATU-8988 and (E) PL-45 cells after treatment with different concentrations of free and encapsulated PIPD by CCK-8 analysis. (F) Live/dead staining of PATU-8988 and PL-45 cells with different treatments. For necrotic cells, the reactive dye can permeate the compromised membranes and react with free amines both in the interior and on the cell surface. In contrast, only the cell-surface amines of viable cells are available to react with the dye, resulting in relatively dim staining. Scale bar: 50 µm. All the data are mean ± SD (n=3), **P<0.01.

Figure 7

A. Changes in mitochondrial membrane potential of PATU-8988 and PL-45 cells treated with free and encapsulated PIPD by CLSM. Scale bar: 20 μm. B. Relative protein expression of PDEδ in PATU-8988 cells after treated with different concentrations of PIPD and CM8988-PIPD for different times by WB. C. Relative protein expression of PDEδ, T-AKT, P-AKT, T-Erk, P-Erk, and Cleaved Caspase 9 in PATU-8988 and PL-45 cells treated with different formulations of PIPD.