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. Author manuscript; available in PMC: 2022 Aug 10.
Published in final edited form as: Eur J Pharm Biopharm. 2020 Jul 6;154:43–49. doi: 10.1016/j.ejpb.2020.06.019

Charge reversible hyaluronic acid-modified dendrimer-based nanoparticles for siMDR-1 and doxorubicin co-delivery

Xin Zhang 1,2, Jiayi Pan 2, Momei Yao 2, Livia Palmerston Mendes 2,3, Can Sarisozen 2, Shirui Mao 1,*, Vladimir P Torchilin 2,4,*
PMCID: PMC9364115  NIHMSID: NIHMS1611108  PMID: 32645383

Abstract

Dendrimer-based nanoparticles have shown promising applications in delivery of small interference RNA (siRNA) to downregulate proteins that contribute to multidrug resistance (MDR). Various types of modification can further enhance the anti-tumor efficacy of dendrimer-based nanoparticles. In this study, generation 4 polyamodoamine (PAMAM) was conjugated with PEG2k-DOPE. The PAMAM-PEG2k-DOPE co-polymer, together with mPEG2k-DOPE, was formulated into mixed dendrimer micelles (MDMs) that can complex siRNA through the cationic PAMAM moieties and encapsulate hydrophobic drug in the micellar lipid cores. DOPE-conjugated hyaluronic acid (HA) was coated on the surface of MDMs to shield the exposed positive charge on PAMAM and to increase the cellular association with CD44+ cancer cells. The HA-modified MDMs could form stable complexes with siRNA, prevent RNase-mediated siRNA degradation and maintain its integrity. Cellular association and cytotoxicity of HA-modified MDMs were investigated in A2780 ADR, MDA-MB-231 and HCT 116 cell lines. The HA-modified MDMs alleviated the toxicity from cationic charge, increase the cancer cell specificity and enhance the cancer cell killing effect in CD44+ cell line.

Keywords: Hyaluronic acid (HA), PAMAM dendrimers, mixed dendrimer micelles (MDMs), multidrug resistance, siMDR-1, doxorubicin base (Dox), co-delivery system

Graphical Abstract

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1. Introduction

Multidrug resistance (MDR) remains one of the unresolved issues for cancer therapy. By attenuating the efficacy of chemotherapy drugs, it contributes a significant impact on therapeutic failure in cancer treatment and on tumor recurrence [1, 2]. There are multiple mechanisms that result in MDR, including increased drug efflux [3, 4], activation of detoxifying systems [5], a malfunctional DNA repair system [6] and evasion of drug-induced apoptosis [7]. Reversal of multidrug resistance by interfering in these cellular pathways has been extensively investigated over the last several decades, especially by inhibiting the function or downregulating the expression of membrane-bound ATP-binding cassette transporters [8]. Overexpression of membrane-bound transporters, such as P-glycoprotein (P-gp) [9] and breast cancer resistance protein (BCRP) [10], leads to an increased efflux of a wide range chemotherapeutic agents in resistant cancers from multiple origins [11]. Delivery of small molecule P-gp inhibitors or small interfering RNA (siRNA) to MDR cancer cells using nanoparticles has been reported to restore the sensitivity to chemotherapeutics [12, 13]. A co-delivery platform for both siRNA and chemotherapeutics provides a synergistic anti-cancer effect in treating MDR cancer [14–16].

The application of siRNA in downregulating the membrane-bound P-gp has attracted great attention because of its high specificity and bio-compatibility. Cationic polymers, including poly(amidoamine) dendrimers (PAMAM), polyethyleneimine(PEI), poly-L-lysine (PLL) and chitosan-based non-viral nanocarriers has been extensively applied to condense and load anionic siRNA molecules by forming complexes through electrostatic interaction [17]. The ‘proton-sponge’ effect, resulting from the amine groups within cationic polymer, facilitates the endosomal escape and the release of siRNA to the cytoplasm [18–20]. However, the cytotoxicity and serum stability of cationic polymers associated with their cationic nature hampers their development [21]. To make up for these deficiency of cationic polymers-based drug carriers, surface shielding of positive charge of cationic polymers using functional anionic polymers can provide an effective approach.

Hyaluronic acid (HA) is a hydrophilic anionic polymer, composed of D-glucuronic acid and N-acetyl-D-glucosamine and found in the synovial fluid and extracellular matrix [22]. It has been widely studied for the delivery of small molecules and nucleic acids in cancer treatment [23]. In addition to excellent biocompatibility and biodegradability, HA has been applied to target overexpressed receptors in cancer cells, such as CD44 receptor, lymphatic vessel endothelial receptor-1 (LYVE-1) and hyaluronic acid-mediated motility [23–25]. When incorporated with cationic gene carriers, the negative charges of HA efficiently neutralize the positive charge of cationic polymers so as to decrease the cytotoxicity and increase the systemic circulation time [26–28].

In a previous reported study [29], mixed dendrimer micelles (MDMs) composed of G4 PAMAM-PEG2K-DOPE was successfully developed as a siRNA/Dox co-delivery nanocarrier and that exhibited promise as an approach to overcome MDR cancer. In this study, charge reversible HA-modified MDM nanoparticles were designed for the co-delivery of MDR-1 siRNA and Dox base (Figure 1). The negatively charged HA coating shielded the exposed cationic charges of dendrimer and provided a targeting property to the CD44 receptors to increase the cellular association in CD44-overexpressed cancer cells. The binding ability of siRNA, the RNAse protection effect and serum stability were studied, and the difference between HA and HA-DOPE was also compared. The cell association, cytotoxicity of MDM, HA/MDM and HA-DOPE/MDM in CD44– A2780 ADR and CD44+ MDA-MB-231 and HCT 116 cell lines were also studied.

Figure 1.

Figure 1.

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Schematic illustration of the charge reversible hyaluronic acid-modified mixed dendrimer micelles for co-delivery of siMDR-1 and doxorubicin base.

2. Material and Method

2.1. Material

Hyaluronic acid (50 kDa) was purchased from Creative PEGWorks (NC, USA). Doxorubicin HCl was purchased from LC Laboratories (MA, USA). ON-TARGET Plus siRNA targeting MDR-1 gene (siMDR-1): 5’-GGAAAAGAAACCAACUGUCdTdT-3’ (sense) were purchased from Horizondiscovery Ltd. (Waterbeach, UK); G4 PAMAM with an ethylenediamine core, (N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride) (EDC) and Sepharose® CL-4B column were purchased from Sigma-Aldrich (MO, USA). 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) (PEG2k-DSPE) was bought from Corden Pharma (MA, USA). pNP-PEG2k-pNP (2 kDa) was purchased from Laysan Bio (AL, USA). 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (Rhodamine-DOPE) were bought from Avanti Polar Lipids (AL, USA). N-hydroxysuccinimide (NHS) was purchased from Thermo Scientific (MA, USA). CellTiter blue® was purchased from Promega (WI, US). A2780 ADR, HCT116, MDA-MB-231 cells were cultured in RPMI with L-glutamine from CellGro (VA, USA) and McCoy’s 5A-modified medium from GIBCO (MD, USA) and DMEM with high glucose from CellGro, respectively. All cells were purchased from The American Type Culture Collection (ATCC).

2.2. Conjugation of PAMAM-PEG2k-DOPE

The PAMAM-PEG2k-DOPE was conjugated according to the method developed by Biswas, et al [29, 30]. Briefly, to preparation of pNP-PEG2k-DOPE, pNP-PEG2k-pNP (MW 2000 Da, 500 mg) and DOPE (38.7 mg) mixed with TEA (23 μl) were reacted overnight at room temperature in anhydrous chloroform. Chloroform was then removed by rotary-evaporation. The crude was resuspended in ultrapure water and purified using a Sepharose® CL-4B column with a 0.0001 M HCl solution.

To prepare PAMAM-PEG2k-DOPE, pNP-PEG2k-DOPE (4.8 mg) and G4 PAMAM (24.9 mg) were dissolved in DMF and reacted overnight at room temperature The DMF was removed by rotary-evaporation and PAMAM-PEG2k-DOPE was purified by dialysis against 50 kDa MWCO regenerated cellulose dialysis membrane. The purified PAMAM-PEG2k-DOPE was lyophilized and dissolved in methanol at 5 mg/ml for future preparation of MDM.

2.3. Synthesis of HA-DOPE

The HA-DOPE was synthesized according to a report by Surace, et al [31, 32]. Briefly, 40 mg of HA was dissolved in distilled water and pre-activated with 7.7 mg EDC and 6.9 mg NHS at pH 7.4 for 3 h at 37 °C. Subsequently, 1.2 mg DOPE (chloroform solution) was dispersed in methanol and added to the HA solution at a volume ratio 4:6. The reaction was kept for 24 h at 37 °C. The conjugates were dialyzed against a 100 kDa MWCO membrane in distilled water for 24 h and then freeze-dried. The HA-DOPE was further purified by washing with chloroform for 3 times to remove free DOPE. The obtained HA-DOPE conjugates were stored at −20 °C for future use.

2.4. Preparation of HA-modified MDM nanoparticles

To prepare HA-modified MDM nanoparticles, Dox-loaded MDM was first prepared according the method reported in a previous study [29]. Briefly, Dox base in methanol, PAMAM-PEG2k-DOPE and PEG2k-DOPE (mole ratio 1:10) were mixed and solvents were evaporated by nitrogen followed by lyophilization. The film was then hydrated with 20 mM buffered HEPE, 5% glucose pH 7.3 (BHG) solution to form MDM and mixed with HA or HA/PE at a volume ratio of 1:4 for 30 mins at 37 °C to form HA/MDM and HA-DOPE/MDM. The ratio between HA and PAMAM-PEG2k-DOPE was further screened and optimized based on size and zeta potential of the obtained nanoparticles.

2.5. Particle size and zeta potential

Particle size and zeta potential of formulation was analyzed by Malvern Zetasizer Nano ZS 90 instrument (Malvern Instruments, Malvern, UK) at 25 °C and scattering angle of 90° based on DLS technology. All measurements were performed in BHG at a total material concentration of 2 mg/ml.

2.6. Stability of siRNA against RNase

Complexation between HA/MDM and HA-DOPE/MDM with siRNA was analyzed by gel electrophoresis. In Accordance with previously reported N/P ratios [30], formulations were mixed and incubated with 0.5 μg siRNA for 30 min at room temperature. Gel electrophoresis was performed in a 2% agarose gel containing ethidium bromide (Invitrogen, MA, USA) at 60 mV for 30 mins. The siRNA bands were visualized under UV light. RNAse protection from complexation was studied by incubating 0.25 U/ml RNase (Ambion, TX, US) with siRNA-complexed formulations (N/P = 4) at equal volume at 37 °C for 30 mins. The activity of RNase was terminated by addition of 25 mM EDTA solution (Sigma). Complexed siRNA was released from formulation by adding 40 μl Heparin (1 U/ml) (American Pharmaceutical Partners, CA, USA). Intact siRNA protected by the complexation was visualized under UV.

N/Pratio=Moles primary amine groups on PAMAM−PEG2k−DOPEMoles of phosphate groups on siMDR−1

2.7. Serum stability

The serum stability of MDM, HA/MDM and HA-DOPE/MDM was evaluated by analyzing the particle size and zeta potential variation before and after incubation with 10% fetal bovine serum (FBS) in PBS at an equal volume at 37 °C for 4 hours. Particle size and zeta potential determination were described in section 2.5.

2.8. Cell association

Cell association of MDM, HA/MDM, HA-DOPE/MDM in A2780 ADR, MDA-MB-231 and HCT116 was evaluated with flow cytometry (Beckton Dickinson FACSCaliburTM, NJ, USA). Cells (80,000/well) were seeded in 12-well plates. After overnight incubation, MDM formulations containing 1 mole% of Rhodamine-DOPE were added to the cells, followed by incubation for 4 hours. After that, cells were collected and washed 3 times with PBS pH 7.4. The fluorescence signal of 10,000 gated living cell events was collected at Ex=488 nm and Em=585/42 nm.

2.9. Expression of CD44

Phycoerythrin-labeled mouse anti-human monoclonal CD44 antibody (clone: G44–26) (Fisher Scientific, MA) was used for membrane-bound CD44 staining. IgG2b, kappa, labeled with phycoerythrin was used as the isotype control. Cells were harvested with enzyme-free cell dissociation buffer to keep the membrane bound CD44 intact. After harvesting, cells were counted and resuspended in blocking buffer containing 2% BSA (w/v) and 0.1% (w/v) NaN3 in PBS, pH 7.4 at a concentration of 106 cells per 100 μl of cell suspension. The antibody was incubated with cells on ice at 5 μl/106 cells for 30 mins before 3x washing with PBS pH 7.4. The fluorescence signal was excited with a 488 nm blue laser using a flow cytometry and recorded with a 585/42 wavelength filter. A total of 10,000 gated living cell events were collected.

2.10. Cytotoxicity

Cytotoxicity of MDM, HA/MDM, HA-DOPE/MDM in A2780 ADR, MDA-MB-231 and HCT 116 cell line were quantified with the CellTiter blue® assay. Briefly, 3000 cells/well were seeded in 96-well plates and incubated overnight. Cells were treated with formulations in serum complete media at multiple concentrations. Media with formulation was replaced with fresh serum complete media after 2 hours’ incubation. The cytotoxicity of formulation was evaluated by incubating with CellTiter blue® at 48 hours after the treatment following the manufacturer’s protocol (Promega, WI, USA).

2.11. Statistical analysis

Data was expressed as mean ±standard deviation (SD) and statistical analysis was performed by Student’s t-test for comparing two groups, one-way ANOVA multiple comparison in cellular association studies and two-way ANOVA multiple comparison in cytotoxicity studies. p ≤ 0.05 was considered as statistically significant.

3. Results and Discussion

3.1. The influence of HA coating on the property of nanoparticles

The toxicity of PAMAM dendrimers comes mainly from their cationic charges, which interact with the negatively charged cell membranes and induce the membrane disruption [33]. Masking the surface charge of PAMAM structure with HA provides an effective way to minimize its toxicity. MDM, composed of G4 PAMAM-PEG2k-DOPE and PEG2k-DSPE, was coated with HA-DOPE lipid-polymer conjugate. Addition of DOPE on HA created a higher hydrophobic interaction, that interacted with the hydrophobic moieties in PAMAM dendrimer and MDM micellar cores to achieve a more stable HA coating. The influence of mole ratio between HA-DOPE and MDM on the properties of nanoparticles is shown in Figure 2. The zeta potential of nanoparticles decreased with the increasing mole ratio between HA-DOPE and MDM nanoparticles consist of PAMAM-PEG2k-DOPE and PEG2k-DSPE. Formulations possessed a negative charge when the mole ratios of HA-DOPE/MDM were above 0.5. The reversal in charge indicated the successful shielding of positive charge on MDM by HA. The HA-DOPE-coated MDM nanoparticles had a larger diameter than those without HA-DOPE coating. This phenomenon could come from the large molecule weight and loose structure of hyaluronic acid, which resulted in a much larger hydrodynamic size observed in dynamic light scattering measurement. With the increasing amount of hyaluronic acid, the size of particles was further elevated because of a thicker layer of hydrodynamically loose coating. At the mole ratio between HA-DOPE and MDM equals to 1, a large particle size around 225 nm and a negative zeta potential around −18 mV were not preferable in consideration of the enhanced permeation and retention effect. Therefore, the mole ratio between HA-DOPE and MDM at 0.5 was selected to prepare the HA-DOPE/MDM. To further understand the influence of the DOPE group in HA on the performance of cellular interaction and cytotoxicity, HA/MDM was also prepared with the same mole amount of HA as that in HA-DOPE/MDM formulations. The result showed that there was no significant difference between HA /MDM and HA-DOPE MDM in particle size and surface charge.

Figure 2.

Figure 2.

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The influence of molar ratio between HA-DOPE coating to MDM nanoparticles on the particle size and zeta potential of HA/MDM and HA-DOPE/MDM. Results indicate mean±SD, n=3.

3.2. The siRNA binding and protection ability of HA-DOPE/MDM

The PAMAM and siRNA complexes formed through electrostatic interaction. However, the reversal of charge in the combined application of HA with cationic drug carrier may affect the binding ability of cationic polymer with anionic nucleic acid molecules, reducing their transfect efficiency, resulting in aggregation of nanoparticles [34–36]. Therefore, the stability of HA-DOPE/MDM/siRNA and HA/MDM/siRNA complexes was investigated (Figure 3a). In both types of coating, siRNA molecules were fully complexed at N/P ratios higher than 0.5. Incorporation of anionic HA did not influence the complexation between PAMAM and siRNA. The high density of the branched PAMAM structure provided binding sites for siRNA to form a stable complex, bypassing the influence from surface modification on the complexation efficiency.

Figure 3.

Figure 3.

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(a) Complexation efficiency of HA-DOPE/MDM and HA/MDM with siRNA at different N/P ratios. (b) Protection effect of siRNA by HA-DOPE/MDM and HA/MDM from RNase-mediated degradation by gel retardation assay at N/P ratio of 4. Free RNase: formulation treated by RNase; Heparin: formulation treated by RNase and then incubated with heparin to release siRNA.

In addition, the ability to protect siRNA from enzyme-mediated degradation is an important characteristics of an ideal delivery system for nucleic acid therapies. In Figure 3b, a faint band from siRNA treated with RNase was observed, indicating the degradation of siRNA. When complexed with HA-DOPE/MDM and HA/MDM, bands of siRNA remained and were released in the presence of heparin. Approximately 85% of siRNA were intact after incubating with RNAse for 30 min. This suggested that the complexation protected the siRNA from RNase-mediated degradation.

3.3. Stability in serum.

The stability of nanoparticles in serum was evaluated to determine their suitability for in vivo applications. When nanoparticles contact biological fluids, protein is rapidly adsorbed on the particle surface to form a corona structure [37, 38]. It will greatly alter the surface property of nanoparticles and result in the stability problems. Stability of formulation in 10% FBS was studied in terms of size and zeta potential. Figure 4 a showed that the size of all the formulations increases after incubating with 10% FBS. The increase of size of positive charged MDM was much higher (5.1 times) than negative HA-DOPE/MDM and HA/MDM (2.2–2.4 times). The zeta potential of MDM also dropped to more negatively charged because of the adsorbed proteins. Surface charge of HA/MDM and HA-DOPE/MDM decreased slightly to about −11 mV. This result indicated that protein adsorption phenomenon of nanoparticle in serum was nonselective and ubiquitous, while HA surface modification could improve the stability and minimize the fluctuation of size and zeta potential.

Figure 4.

Figure 4.

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Stability of MDM, HA/MDM and HA-DOPE/MDM in 10% FBS. (a) Z-average of MDM, HA/MDM and HA-DOPE/MDM nanoparticles before and after incubating with 10% FBS for 4 h. (b) Zeta potential of MDM, HA/MDM and HA-DOPE/MDM after incubating with 10% FBS for 4 h. Results indicate mean ±SD, n=3. ****p ≤ 0.0001, ***p ≤ 0.001, **p ≤ 0.01.

3.4. The pH-responsive charge reversal in HA-modified MDM

The buffering capacity, also known as the “proton sponge” effect, of PAMAM can facilitate the endosomal escape of delivered cargos [39]. To understand whether the surface modification will affect the buffering capacity of PAMAM, the surface charge of HA and HA-DOPE/MDM in different pH environments was studied. In Figure 5, a pH-dependent charge reversing property was found in both MDM formulations coated with HA and HA-DOPE. The pH-responsive of NH3+ group density in PAMAM and COO− group density in HA contribute to the reversal of charge in nanoparticles. When the environment pH turned acidic, the decreasing anionic charge in HA and increasing cationic charge of PAMAM leads to the increase of overall zeta potential of HA-modified MDM. In addition, the weakened electrostatic interactions between HA and PAMAM structure may further induce the HA shedding from the surface. This indicated that HA modified MDM carriers won’t affect the buffering capacity of PAMAM and will still maintain the endosome escape ability of PAMAM.

Figure 5.

Figure 5.

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The charge reversal of HA-DOPE/MDM and HA/MDM in different pH environment. Results indicate mean ±SD, n=3.

3.5. Cytotoxicity of HA-modified MDM

The generation, size and number of amine groups on the surface of PAMAM play crucial roles in determining the cytotoxicity outcomes. The cytotoxicity of plain MDM formulations shielded with HA was evaluated in A2780 ADR, MDA-MB-231 and HCT 116 cell lines (Figure 6). MDM induced a concentration-dependent cytotoxicity. Surface modification with HA and HA-DOPE alleviated the cytotoxicity in all three cell lines due to the anionic charge of HA shielding the positive surface charge on PAMAM. In the MDA-MB-231 cell line, the HA/MDM nanoparticles showed a lower cytotoxicity than the one in HA-DOPE/MDM at high concentration. This might result from the complete coating of HA on the surface of MDM based on electrostatic interactions and prevented the interactions between amine groups of PAMAM and cell membrane. The introduction of DOPE moieties in HA integrated it more tightly with the MDM structure. Thereby, some of the NH3+ groups could be exposed on the surface (Figure 1). The strategies to alleviate toxicity of PAMAM based on covalent bonds modification usually change the properties of PAMAM, which may affect the cell association of nanoparticles. To further understand whether the HA modification based on ionic bonds will also affect their cell association ability, the cell association of formulation in A2780 ADR, HCT 116, MDA-MB-231 cell line was also studied.

Figure 6.

Figure 6.

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Cytotoxicity of MDM, HA-DOPE/MDM, HA/MDM blank carrier for (a) A2780 ADR; (b) MDA-MB-231; (c) HCT 116 cell lines. Results indicate mean±SD, n=3. ****p ≤ 0.0001, ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.5.

3.6. Cellular association

The cell association of PAMAM-based nanoparticles is in a lack of selectivity. Nanoparticles-coated with HA have the potential to actively target cancer cells that overexpress CD44 receptors [40]. Therefore, a HA coating on MDM is expected to enhance the cellular association in CD44+ cancer cells. In Figure 7, multiple cell lines were characterized for their membrane-bound CD44 expression and applied in the evaluation of HA-mediated cellular association. The results showed that the cellular association in A2780 ADR, which exhibited low CD44 expression, was highly dependent on the surface charge of formulation. The positively charged MDM showed the highest cellular association result. For HA-DOPE/MDM and HA/MDM, a decreased cellular association in the A2780 ADR cell line might result from fewer electrostatic interactions between HA-coated nanoparticles and cell membranes. In contrast, the cellular association ability of HA/MDM and HA-DOPE/MDM increased compared to that of MDM in CD44+ cell lines. Additionally, HA-DOPE/MDM showed a higher cellular association effect than HA/MDM and MDM in both CD44-expressing MDA-MB-231 and HCT 116 cell lines. This result also indicated that the introduction of hydrophobic moiety facilitated fusion of HA-DOPE into the MDM nanoparticles and contributed to the CD44-dependent increased cellular association.

Figure 7.

Figure 7.

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Expression of membrane-bound CD44 in different types of cancer cell lines. Results indicate the fluorescence increase compared to the isotype control (a). Cellular association of MDM and HA-coated MDM formulations labelled with 1 mole% Rhodamine-DOPE in (b) A2780 ADR; in (c) HCT 116; in (d) MDA-MB-231 cells. Results indicate mean ±SD, n=3. ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.5.

3.7. Cytotoxicity of HA-modified MDM co-delivery system

The cytotoxicity profiles of MDM loaded with Dox (MDM-D), HA-DOPE-coated MDM-loaded with Dox (HA-DOPE/MDM-D) and HA-DOPE-coated MDM-loaded with both Dox and siMDR-1 (HA-DOPE/MDM-DR) in A2780 ADR, HCT 116 and MDA-MB-231 cell line were studied (Figure 8). The cytotoxicity of formulations in all three cell lines was Dox concentration-dependent and related to the expression of CD44. HA-DOPE/MDM-D-loaded with 2.5 μM or 10 μM of Dox showed higher cytotoxicity compared to MDM-D without HA-DOPE modification in HCT116 cell line. A similar enhancement was observed in MDA-MB-231 cells treated with HA-DOPE/MDM-D and MDM-D loaded with 10 μM Dox. Such HA-mediated cytotoxicity enhancement was not observed in the A2780 ADR cell line due to a significantly lower CD44 expression compared to MDA-MB-231 or HCT116. Both MDA-MB-231 and A2780 ADR had an overexpression of membrane-bound P-gp [30, 41]. With the co-delivery of Dox and siMDR-1, HA-DOPE/MDM-DR resulted in a higher cytotoxicity in MDA-MB-231-treated with 10 μM Dox/100 nM siMDR-1 and A2780 ADR-treated with 2.5 μM Dox/100 nM siMDR-1 or 10 μM Dox/100 nM siMDR-1 because of a combinational effect. However, the combination effect of Dox and siMDR-1 was not significant at a high concentration of Dox in MDA-MB-231 because the cytotoxicity from Dox masked the combination effect from co-delivery. With a low membrane-bound P-gp expression, the combination of Dox and siMDR-1 delivered by HA-DOPE/MDM-DR showed a similar cytotoxicity to HA-DOPE/MDM-D without siMDR-1 at both Dox concentrations in the HCT116 cell line. Therefore, HA-DOPE/MDM-D significantly enhanced the cytotoxicity in CD44-expressing cell lines. The co-delivery of Dox and siRNA with HA-DOPE-modified MDM can further increase the cytotoxicity and specificity in tumor cells that show overexpression in both membrane-bound CD44 and P-gp proteins.

Figure 8.

Figure 8.

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Cytotoxicity of Dox-loaded MDM, HA-DOPE/MDM-loaded with Dox and HA-DOPE/MDM-loaded with Dox and 100 nM of siMDR-1 in (a) MDA-MB-231; (b) A2780 ADR; (c) HCT 116 cells compared with cells treated with BHG. Results indicate mean±SD, n=3. ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.5.

4. Conclusion

The toxicity and non-selective cell membrane interaction of PAMAM-based nanoparticles may limit their application in drug delivery. Charge-reversible CD44 receptor-targeting HA-DOPE/MDM and HA/MDM were successfully prepared between HA and PAMAM dendrimer-based mixed dendrimer micelles through electrostatic interactions. Results showed that HA modification did not affect the formulation’s complexation ability with siMDR-1. Additionally, it prevented the RNase-mediated degradation of siMDR-1. The negative surface charge induced by HA modification increased the serum stability of MDM and decreased the cytotoxicity coming from the cationic charges on PAMAM structure. Also, the HA-modified MDM structure showed a pH-dependent charge reversal property, which favored the endosomal escape. The CD44-dependent cellular association of HA-modified nanocarriers was observed. With a more compact structure, HA-DOPE/MDM exhibited a better cellular association enhancement compared to that with HA/MDM. The combinational cytotoxicity enhancement in HA-DOPE-modified MDM-loaded with both Dox and siMDR-1 suggests a promising tumor specificity and therapeutic performance for treatment of CD44+ multidrug resistant tumors.

5. Acknowledgement

The authors would like to thank Dr. William Hartner for helpful suggestions on the preparation of manuscript. This work was supported by the National Institutes of Health (NIH) grant [R01CA200844] and was supported by China Scholarship Council (CSC) scholarship to Xin Zhang.

Footnotes

6.

Declaration of interest

None.

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7. Reference

  • 1.Kunjachan S, et al. , Multidrug resistance: Physiological principles and nanomedical solutions. Adv Drug Deliv Rev, 2013. 65(13–14): p. 1852–1865. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Patel NR, et al. , Nanopreparations to overcome multidrug resistance in cancer. Adv Drug Deliv Rev, 2013. 65(13–14): p. 1748–62. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Bradshaw DM and Arceci RJ, Clinical relevance of transmembrane drug efflux as a mechanism of multidrug resistance. J Clin Oncol, 1998. 16(11): p. 3674–90. [DOI] [PubMed] [Google Scholar]
  • 4.Gottesman MM, Fojo T, and Bates SE, Multidrug resistance in cancer: role of ATP-dependent transporters. Nat Rev Cancer, 2002. 2(1): p. 48–58. [DOI] [PubMed] [Google Scholar]
  • 5.O’Brien ML and Tew KD, Glutathione and related enzymes in multidrug resistance. Eur J Cancer, 1996. 32A(6): p. 967–78. [DOI] [PubMed] [Google Scholar]
  • 6.Fink D, Aebi S, and Howell SB, The role of DNA mismatch repair in drug resistance. Clin Cancer Res, 1998. 4(1): p. 1–6. [PubMed] [Google Scholar]
  • 7.Tsuruo T, et al. , Molecular targeting therapy of cancer: drug resistance, apoptosis and survival signal. Cancer Sci, 2003. 94(1): p. 15–21. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Chen Z, et al. , Mammalian drug efflux transporters of the ATP binding cassette (ABC) family in multidrug resistance: A review of the past decade. Cancer Lett, 2016. 370(1): p. 153–64. [DOI] [PubMed] [Google Scholar]
  • 9.Gottesman MM, Pastan I, and Ambudkar SV, P-glycoprotein and multidrug resistance. Curr Opin Genet Dev, 1996. 6(5): p. 610–7. [DOI] [PubMed] [Google Scholar]
  • 10.Staud F and Pavek P, Breast cancer resistance protein (BCRP/ABCG2). Int J Biochem Cell Biol, 2005. 37(4): p. 720–5. [DOI] [PubMed] [Google Scholar]
  • 11.Ganoth A, Merimi KC, and Peer D, Overcoming multidrug resistance with nanomedicines. Expert Opin Drug Deliv, 2015. 12(2): p. 223–38. [DOI] [PubMed] [Google Scholar]
  • 12.Zhang Y, et al. , Reversal of Chemoresistance in Ovarian Cancer by Co-Delivery of a P-Glycoprotein Inhibitor and Paclitaxel in a Liposomal Platform. Mol Cancer Ther, 2016. 15(10): p. 2282–2293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Navarro G, et al. , P-glycoprotein silencing with siRNA delivered by DOPE-modified PEI overcomes doxorubicin resistance in breast cancer cells. Nanomedicine (Lond), 2012. 7(1): p. 65–78. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Essex S, et al. , Phospholipid-modified PEI-based nanocarriers for in vivo siRNA therapeutics against multidrug-resistant tumors. Gene Ther, 2015. 22(3): p. 257–266. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Szakacs G, et al. , Targeting multidrug resistance in cancer. Nat Rev Drug Discov, 2006. 5(3): p. 219–34. [DOI] [PubMed] [Google Scholar]
  • 16.Xiong XB and Lavasanifar A, Traceable multifunctional micellar nanocarriers for cancer-targeted co-delivery of MDR-1 siRNA and doxorubicin. ACS Nano, 2011. 5(6): p. 5202–13. [DOI] [PubMed] [Google Scholar]
  • 17.Sarisozen C, et al. , Polymers in the co-delivery of siRNA and anticancer drugs to treat multidrug-resistant tumors. Journal of Pharmaceutical Investigation, 2017. 47(1): p. 37–49. [Google Scholar]
  • 18.Boussif O, et al. , A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proceedings of the National Academy of Sciences, 1995. 92(16): p. 7297–7301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Pack DW, et al. , Design and development of polymers for gene delivery. Nat Rev Drug Discov, 2005. 4(7): p. 581–93. [DOI] [PubMed] [Google Scholar]
  • 20.Varkouhi AK, et al. , Endosomal escape pathways for delivery of biologicals. J Control Release, 2011. 151(3): p. 220–8. [DOI] [PubMed] [Google Scholar]
  • 21.Kircheis R, Wightman L, and Wagner E, Design and gene delivery activity of modified polyethylenimines. Advanced drug delivery reviews, 2001. 53(3): p. 341–358. [DOI] [PubMed] [Google Scholar]
  • 22.Burdick JA and Prestwich GD, Hyaluronic acid hydrogels for biomedical applications. Adv Mater, 2011. 23(12): p. H41–56. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Dosio F, et al. , Hyaluronic acid for anticancer drug and nucleic acid delivery. Adv Drug Deliv Rev, 2016. 97: p. 204–36. [DOI] [PubMed] [Google Scholar]
  • 24.Dufay Wojcicki A, et al. , Hyaluronic acid-bearing lipoplexes: physico-chemical characterization and in vitro targeting of the CD44 receptor. J Control Release, 2012. 162(3): p. 545–52. [DOI] [PubMed] [Google Scholar]
  • 25.Mattheolabakis G, et al. , Hyaluronic acid targeting of CD44 for cancer therapy: from receptor biology to nanomedicine. J Drug Target, 2015. 23(7–8): p. 605–18. [DOI] [PubMed] [Google Scholar]
  • 26.Kesharwani P, et al. , Hyaluronic acid-conjugated polyamidoamine dendrimers for targeted delivery of 3, 4-difluorobenzylidene curcumin to CD44 overexpressing pancreatic cancer cells. Colloids and Surfaces B: Biointerfaces, 2015. 136: p. 413–423. [DOI] [PubMed] [Google Scholar]
  • 27.Yang X, et al. , MDR1 siRNA loaded hyaluronic acid-based CD44 targeted nanoparticle systems circumvent paclitaxel resistance in ovarian cancer. Sci Rep, 2015. 5: p. 8509. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Qi X, et al. , Hyaluronic acid-grafted polyamidoamine dendrimers enable long circulation and active tumor targeting simultaneously. Carbohydr Polym, 2015. 126: p. 231–9. [DOI] [PubMed] [Google Scholar]
  • 29.Biswas S, et al. , Lipid modified triblock PAMAM-based nanocarriers for siRNA drug co-delivery. Biomaterials, 2013. 34(4): p. 1289–301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Pan J, et al. , Polyamidoamine dendrimers-based nanomedicine for combination therapy with siRNA and chemotherapeutics to overcome multidrug resistance. Eur J Pharm Biopharm, 2019. 136: p. 18–28. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Surace C, et al. , Lipoplexes targeting the CD44 hyaluronic acid receptor for efficient transfection of breast cancer cells. Mol Pharm, 2009. 6(4): p. 1062–73. [DOI] [PubMed] [Google Scholar]
  • 32.Wojcicki AD, et al. , Hyaluronic acid-bearing lipoplexes: physico-chemical characterization and in vitro targeting of the CD44 receptor. Journal of controlled release, 2012. 162(3): p. 545–552. [DOI] [PubMed] [Google Scholar]
  • 33.Jain K, et al. , Dendrimer toxicity: Let’s meet the challenge. Int J Pharm, 2010. 394(1–2): p. 122–42. [DOI] [PubMed] [Google Scholar]
  • 34.Jevprasesphant R, et al. , The influence of surface modification on the cytotoxicity of PAMAM dendrimers. Int J Pharm, 2003. 252(1–2): p. 263–6. [DOI] [PubMed] [Google Scholar]
  • 35.Kolhatkar RB, et al. , Surface acetylation of polyamidoamine (PAMAM) dendrimers decreases cytotoxicity while maintaining membrane permeability. Bioconjug Chem, 2007. 18(6): p. 2054–60. [DOI] [PubMed] [Google Scholar]
  • 36.Nam HY, et al. , Biodegradable PAMAM ester for enhanced transfection efficiency with low cytotoxicity. Biomaterials, 2009. 30(4): p. 665–73. [DOI] [PubMed] [Google Scholar]
  • 37.Diaz C, et al. , Cytotoxicity and in vivo plasma kinetic behavior of surface-functionalized PAMAM dendrimers. Nanomedicine, 2018. 14(7): p. 2227–2234. [DOI] [PubMed] [Google Scholar]
  • 38.Lundqvist M, et al. , Nanoparticle size and surface properties determine the protein corona with possible implications for biological impacts. Proc Natl Acad Sci U S A, 2008. 105(38): p. 14265–70. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Dufes C, Uchegbu IF, and Schatzlein AG, Dendrimers in gene delivery. Adv Drug Deliv Rev, 2005. 57(15): p. 2177–202. [DOI] [PubMed] [Google Scholar]
  • 40.Huang WY, et al. , Nanoparticle Targeting CD44-Positive Cancer Cells for Site-Specific Drug Delivery in Prostate Cancer Therapy. ACS Appl Mater Interfaces, 2016. 8(45): p. 30722–30734. [DOI] [PubMed] [Google Scholar]
  • 41.Chen J, et al. , PKD2 mediates multi-drug resistance in breast cancer cells through modulation of P-glycoprotein expression. Cancer Lett, 2011. 300(1): p. 48–56. [DOI] [PubMed] [Google Scholar]

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