Host polymer for siRNA condensation and cytoplasmic delivery
The host-guest screening system is composed of the CD-SS-P host polymer for siRNA condensation and endosomal escape, and switchable guest polymers for PNP shielding and targeting. The redox-sensitive CD-SS-P with multiple arms of pDMAEMA linked to the β-CD core by disulfide bonds was synthesized using the method in our previous report (25). The control polymer CD-P, where multiple arms of pDMAEMA were linked to the β-CD core without disulfide bonds, was synthesized too (refer to the Supplementary Materials). The 1H nuclear magnetic resonance (NMR) spectrum of CD-SS-P is shown in Fig. 2A. The β-CD core was grafted with four arms of pDMAEMA. The average degree of polymerization of each pDMAEMA arm was 12. A single peak was observed in the gel permeation chromatography (GPC) elution curve of CD-SS-P (Fig. 2B). The molecular weight was determined to be 7.8 kDa with PDI (polydispersity index) of 1.15. The CD-SS-P was able to completely condense siRNA at an N/P ratio of 3 and above (Fig. 2C). We selected the redox-sensitive CD-SS-P as the host polymer component for the screening platform for the following reasons: (i) The pDMAEMA arms can condense siRNA efficiently and assist endosomal escape of PNPs induced by the “proton sponge” effect; (ii) the β-CD core can allow host-guest complexation of different guest polymers (PEG with different architectures and ligand density) to form the PNP shells; (iii) the disulfide bonds can be cleaved by the intracellular glutathione to readily release siRNA into cytoplasm; and (iv) the redox-sensitive biodegradability of CD-SS-P makes the polymer very low cytotoxic (25). To confirm that CD-SS-P can deliver siRNA into cells and readily release siRNA in cytoplasm, both CD-SS-P and CD-P were labeled by rhodamin-B-isothiocyanate (RITC) via the hydroxyl groups of β-CD and used to deliver siRNA labeled by fluorescein isothiocyanate (FITC) in cultured KB cells (Fig. 2D). The spatial confocal microscopic images showed that green dots (FITC-siRNA) and red dots (RITC-CD-SS-P) were observed separately after internalization (1, 2, and 24 hours after incubation), indicating that most of the siRNA was released from the CD-SS-P carrier (Fig. 2D, upper panel). However, the control CD-P without disulfide linkages failed to release the siRNA in cytosol, because most of the dots were yellow, which are the colocalized FITC-siRNA and RITC-CD-P carrier (Fig. 2D, bottom panel).
Screening of PEG architectures and ligand densities
To obtain an efficient siRNA targeted delivery system, nonspecific binding to cells must be first minimized, as the positively charged gene vehicle tends to bind to the surfaces of all cells (30, 31). PEGylation shielding is the predominant method to minimize the nonspecific binding. Once minimal nonspecific binding is achieved with proper architectures of PEG, the number and density of the ligands at the distal ends of PEG can be optimized. To this end, we assembled the CD-SS-P host polymer with a series of Ad-PEG guest polymers having linear and comb-shaped structures and different sizes, without or with distal ligands (Fig. 3, A and B), to screen and find out the best combinations for the most efficient siRNA targeted delivery.
First, linear Ad-PEG (PL) with Mn of 4k, 8k, 12k, and 20k (PL4k, PL8k, PL12k, and PL20k), and comb-shaped Ad-PEG (PC) with Mn of 4k, 8k, 12k, and 20k (PC4k, PC8k, PC 12k, and PC20k) were synthesized (fig. S1) and screened as shielding polymers for the siRNA PNPs for achieving the minimized nonspecific binding to nontarget cells. By combining the CD-SS-P host and the above Ad-PEG guest polymers, a series of host-guest siRNA gene carriers (shortened as H/G followed by the name of the guest used in a parenthesis) were obtained. The particle size and zeta potential of the siRNA-loaded PNPs formed by the H/G carriers were measured (Fig. 3C). For both linear and comb-shaped Ad-PEG guests, with the increase in PEG molecular weight, the PNP particle size increased from around 75 to 230 nm, whereas the zeta potential decreased from around 32 to below 10 mV. With similar molecular weights, comb-shaped Ad-PEG was more efficient to reduce the zeta potential than linear Ad-PEG. The H/G(PL20k), H/G(PC8k), H/G(PC12k), and H/G(PC20k) carriers gave very low zeta potentials (<10 mV) at an N/P ratio of 20, while their particle size remained within a suitable range for gene delivery (<250 nm). Gene delivery PNPs with low zeta potentials (<10 mV) may have minimal nonspecific binding and prolonged in vivo circulation (32).
To evaluate the H/G carriers for nonspecific binding, FITC-siRNA–loaded PNPs formed by the H/G carriers were cultured with two cell lines, the KB (FR overexpressed, FR+) and A549 (FR deficient, FR−) cells. The cell-associated mean fluorescence intensity (MFI) at 4 hours after transfection was measured to determine the shielding effects of the PEG shells with different sizes and architectures (Fig. 3D). The CD-SS-P host polymer (H) alone without PEG shielding showed very high MFI values in both cell lines, indicating a very high nonspecific binding. H/G(PL20k), H/G(PC8k), H/G(PC12k), and H/G(PC20k) exhibited significantly lower MFI values than other carriers in both cell lines. Linear PEG with longer lengths showed a stronger shielding effect than those with shorter ones. As compared with linear PEG, comb-shaped PEG with similar molecular weight displayed greater shielding power. However, it is noted that longer PEG led to larger particle sizes (Fig. 3C), which may affect the pharmacokinetics of the PNPs (19). On the basis of the results, for linear Ad-PEG, PL20k showed the best effect on PNP shielding. For comb-shaped Ad-PEG, PC8k, PC12k, and PC20k showed the best effect on PNP shielding, whereas PC8k gave smaller particle size than PC12k and PC20k. Therefore, the linear PL20k and the comb-shaped PC8k were chosen for further screening and optimizing of the overall effects of nonspecific binding and ligand-induced siRNA targeted delivery.
Next, FA as the targeting ligand was conjugated to the distal end(s) of PL20k and PC8k. For PL20k, there was only one –OH end, so one FA ligand was conjugated to give PL20k-FA1 (PL20kF1) (fig. S1). For PC8k, there were multiple –OH ends, so 1, 3, 6, 9, and 12 FA ligands were conjugated, to give a series of PC8k-FAn (n = 1, 3, 6, 9, and 12) (PC8kFn) (Fig. 3B). The CD-SS-P host and the six guests (one PL20kF1 and five PC8kFn) were used to form six H/G self-assembled siRNA targeted delivery carriers, and FR+ KB cells were used to evaluate the siRNA-loaded H/G carriers for their specific binding to the FRs on the cells, to screen and find out the optimized combinations of the PEG architecture and the FA ligand density of the H/G carriers.
Binding affinity and dissociation kinetics are two key parameters to determine the specific binding of the H/G carriers to the surface of the KB cells (31, 33). The binding affinity of H/G targeted delivery carriers to KB cells was analyzed by comparing the specific binding and nonspecific binding. FITC-siRNA–loaded H/G carriers with FA ligand (specific binding) and corresponding H/G carriers without ligand (nonspecific binding) were incubated with KB cells respectively on ice for 1 hour, and the fluorescence intensity of the cells was measured after washing out the unbound PNPs. The difference of the fluorescence intensity of the cells treated by H/G with FA and without FA, which represents the net specific binging, was plotted against the siRNA concentration (Fig. 3E). Among six H/G targeted delivery carrier systems, the fluorescence of H/G(PC8kF6) increased much more than other carriers with an increase in siRNA concentration (i.e., PNP concentration), indicating that its PNPs had the strongest specific binding to KB cells. The measured EC50 (the concentration leading to 50% binding) for H/G(PC8kF6) was the lowest among all formulations. The EC50 value was approximately 10-fold lower than those of the monovalent ligand formulations H/G(PL20kF1) and H/G(PC8kF1).
For investigation of the dissociation kinetics, a dissociation binding experiment was conducted. After incubation of FITC-siRNA–loaded PNPs (2000 μmol/ml) in the KB cells for 4 hours, the cells were washed to allow the bound PNPs to dissociate from the surfaces of the cells, while the fluorescence intensity of the cells with remaining bound PNPs were measured and plotted against time (Fig. 3F). The curves showed that among six H/G targeted carriers, the longest dissociation time was needed for the H/G(PC8kF6) delivery system, which is in line with the result that the PNPs formed by H/G(PC8kF6) had the strongest specific binding to KB cells.
It was reported that FR exists as “receptor clusters” in the membrane of KB cells (20, 22, 34, 35). Our results showed that carriers with low FA densities failed to achieve a high binding avidity between the carriers and the cell surfaces. For example, H/G(PL20kF1) and H/G(PC8kF1) only had weak monovalent interaction with the receptor clusters. The binding avidity increased with the ligand density, because higher ligand density allowed multivalent interaction between the carriers and the receptor clusters. When FA density increased beyond optimal values, the binding avidity decreased. Overcrowding may possibly prevent ligands from optimal orientation and spatial arrangement for efficient binding (19). Also, competitions may occur between multiple ligands for a single receptor, which tends to limit the access of ligands to receptors. Several recent publications also shared similar observations with ours (19, 20, 30, 31).
The above results indicate that, for the specific case of KB cells, the intermediate FA ligand density together with the size and architecture of the comb-shaped PEG in the H/G(PC8kF6) carrier led to the optimal specific binding to the target KB cells, whereas the nonspecific binding to nontarget cells was efficiently minimized. It was also confirmed that the H/G(PC8kF6)-based PNPs showed undetectable cytotoxicity at an N/P ratio up to 50 (fig. S2A) and excellent capacity against protein adsorption (fig. S2B). Thus, the formulation based on H/G(PC8kF6) was identified to be the optimized H/G targeted delivery carrier for the following siRNA delivery and gene silencing studies.
In vivo therapeutic efficacy of siRNA-Bcl2 targeted delivery in KB xenograft tumor model
To further prove the optimized H/G(PC8kF6) carrier could function efficiently in vivo as a shielding and targeted delivery system, the siRNA-Bcl2–loaded H/G(PC8kF6) PNPs were evaluated in a mouse KB xenograft tumor model. Phosphate-buffered saline (PBS), H, and H/G(PC8k) treatment groups were also tested as controls. Anticancer efficacy and safety of PNPs were evaluated by tumor volume changes, body weight change, survival rate, hematological and urinal test, tumor histological analysis, and immunofluorescence of Bcl-2. Male BALB/c nude mice were inoculated with KB cells in the right flank. When the tumors reached 50 to 100 mm3 on day 10 after the inoculation, PNPs loaded with siRNA-Bcl2 at a dose of 1000 pmol/kg were injected via the tail vein every 2 days until day 20 after the first injection (Fig. 6A). Tumor growth and body weight of the mice were measured every 2 days. A pronounced inhibition in tumor growth was observed for the H/G(PC8kF6) group (Fig. 6B). From days 4 to 20, the tumors for the control groups increased for 9- to 22-fold in volume, whereas the growth of tumors for the H/G(PC8kF6) group was significantly suppressed. The tumor volume of the H/G(PC8k) group was suppressed to ~0.14-fold of PBS group, which is more efficient than other Bcl-2–siRNA targeted delivery systems reported (38). It must be noted that the antitumor capacity of H/G(PC8k) was also significantly improved although it was not as good as H/G(PC8kF6), probably due to the stability of particles, which would lead to long in vivo circulation and kill cancer via enhanced permeability and retention (EPR) effect. It was beneficial from the optimized length and architecture of PEG as corona of polyplexes. In addition to long circulation and passive targeting, active targeting of H/G(PC8kF6) through receptor-ligand interaction further improved the anticancer efficiency by tumor retention. The body weight was steady or slightly increased for the H/G(PC8kF6) group, showing that the PNPs were safe, while other groups also maintained the body weight during the 21-day treatment period (Fig. 6C). Images of the KB xenograft tumors of the mice at days 0 and 10 and day 20 (or experimental end point) are shown in fig. S4. Figure 6D shows the images of the tumors from various treatment groups at the experimental end point. A 100% survival rate up to 21 days after treatment was observed for the KB tumor–bearing mice treated with PNPs of H/G(PC8kF6), as compared with 35 to 85% survival rates for those treated by other control formulations (Fig. 6E). The ex vivo histological analyses further confirmed that the formulations showed no acute toxicity in the liver, kidney, heart, spleen, and lung (fig. S5). The hematology results indicate that all measured factors were within normal ranges (39), suggesting that no inflammatory reaction was associated with the treatments for all groups, and no liver injury was found (fig. S6A). Urine samples were examined and confirmed that kidney functions were not affected by the PNP treatment (fig. S6B).
Hematoxylin and eosin (H&E) examination of the tumor sections revealed that the tumors treated with siRNA-Bcl2–loaded H/G(PC8kF6) had extensive granulation and showed evidence of necrosis (fig. S7A, upper panel). TUNEL (terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling) staining showed extensive apoptosis in the tumors of the H/G(PC8kF6) group (fig. S7B, lower panel). In addition, immunofluorescence staining for Bcl-2 showed that green fluorescence signals of the H/G(PC8kF6) group was much weaker than those of the other control groups (fig. S7B). The results indicate that the H/G(PC8kF6) group significantly decreased Bcl-2 expression, which benefited the apoptosis of tumor cells.
Whole-body and ex vivo fluorescence imaging
Qualitative nanoparticle biodistribution was studied to evaluate the tumorous targeting and retention of PNPs of H/G(PC8kF6) loaded with Cy5-labeled siRNA (Cy5-siRNA). PBS, H, and H/G(PC8k) were used as control groups. Whole-body fluorescence distribution and organ distribution (tumor, liver, kidney, heart, and lung) of nanoparticles at various time points were evaluated. KB tumor–bearing mice were intravenously injected with the PNPs via the tail vein at a single dose of 1000 pmol/kg of Cy5-siRNA, followed by imaging at different time points through measuring fluorescence signal to capture the whole-body distribution pattern (Fig. 6F). The fluorescence signals of siRNA in the H/G(PC8kF6) group started to accumulate early at 2 hours in the tumor area (red circle) and showed the strongest signal at 24 hours (Fig. 6F). The MFI of Cy5-siRNA in the tumor area for the H/G(PC8kF6) group was significantly higher than those of all other control groups at 2, 8, and 24 hours (fig. S7C). The results indicate that H/G(PC8kF6) PNPs exhibited prolonged circulation and tumor accumulation properties. Because of the limited penetration depth of the light source through the tissue in the whole-body imaging, a different set of mice were euthanized, and their tumors and major organs (liver, kidney, heart, and lung) were collected for ex vivo imaging (Fig. 6G). In fig. S7 (D to G), quantitative fluorescence intensity analyses confirmed that the H/G(PC8k) and H/G(PC8kF6) groups, both with appropriate PEG shielding, showed much lower accumulation in the liver and kidney at the first 24 hours, as compared with the polyethylenimine (PEI) and H groups (both without PEG shell). The excised tumors showed a stronger signal at 2, 8, and 24 hours after injection for H/G(PC8kF6) PNPs, as compared with all the other control groups, indicating that the optimized PC8kF6 shell could enhance the tumor accumulation and retention of the PNPs. It is noted that H/G(PC8kF6) also accumulated in the lung at the 8-hour time point. It was probably due to the lung tissue also expressing some level of FRs (40). Both the therapeutic efficacy and biodistribution results strongly support that H/G(PC8kF6) exhibited long blood circulation and efficient tumor accumulation properties and could successfully deliver siRNA-Bcl2 to tumor cells to silence Bcl-2 protein expression and induce tumor cell apoptosis, resulting in efficient tumor growth suppression.