Engineering a facile and versatile nanoplatform to facilitate the delivery of multiple agents for targeted breast cancer chemo-immunotherapy

doi:10.21203/rs.3.rs-2447818/v1

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Engineering a facile and versatile nanoplatform to facilitate the delivery of multiple agents for targeted breast cancer chemo-immunotherapy

https://doi.org/10.21203/rs.3.rs-2447818/v1

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Background

There is growing evidence showing that single administration of immunotherapeutic agents has limited efficacy in a number of cancer patients mainly due to tumor heterogeneity and immunosuppressive tumor microenvironment. In this study, a novel nanoparticle-based strategy was applied to achieve efficient tumor-targeted therapy by combining chemotherapeutic agents, i.e., doxorubicin (Dox) and melittin (Mel), with an immune checkpoint inhibitor (PD-L1 DsiRNA). The proposed nanoparticle was prepared by the formation of a complex between Mel and PD-L1 DsiRNA (Dicer-substrate short-interfering RNA), followed by the loading of Dox. The surface of the resultant particles (DoxMel/PD-L1 DsiRNA) was then modified with hyaluronic acid (HA) to increase their stability and distribution. In addition, HA can also act as a tumor-targeting agent through binding to its receptor CD44 on the surface of cancer cells.

Results

We demonstrated that the surface engineering of DoxMel/PD-L1 DsiRNA with HA significantly enhances its specificity towards breast cancer cells. Moreover, we observed a noticeable reduction in PD-L1 expression together with a synergistic effect of Dox and Mel on killing cancer cells and inducing immunogenic cell death, leading to significantly diminished tumor growth in 4T1-breast tumor bearing Balb/c mice, improved survival rate and extensive infiltration of immune cells including cytotoxic T cells into the tumor microenvironment. Safety analysis revealed that there is no significant toxicity associated with the developed nanoparticle.

Conclusion

All in all, the proposed targeted combination treatment strategy can be considered as a useful method to reduce cancer-associated mortality.

Cancer immunotherapy

cancer chemotherapy

nanoparticle

targeted delivery

breast cancer

Breast cancer is the most frequently diagnosed malignancy in women globally and is considered the leading cause of cancer-associated death among them [1, 2]. Compared to other types of breast cancer, patients with triple-negative breast cancer (TNBC) show a lower survival rate with a higher risk of recurrence and metastasis [3]. Owing to the lack of estrogen or progesterone receptors and/or human epidermal growth factor receptor 2 (HER-2) in tumor cells, chemotherapy, surgery, and radiotherapy are the main treatments for patients with TNBC [4]. Despite their ability in killing cancer cells, chemotherapeutic drugs cause systemic toxicity and severe side-effects, which significantly restrict their clinical usage [5]. Moreover, many tumors show resistance to different chemotherapeutic drugs [6]. As a result, to achieve a better outcome, it is vital to combine chemotherapy with other novel therapeutic strategies.

Recently, nanotechnology has attracted tremendous attention with diverse applications in medicine. Not only do nanoparticle-based platforms provide opportunities for combining different treatment strategies such as chemotherapy and immunotherapy, but also they are able to simultaneously deliver various payloads to the pre-determined regions with high selectivity [13–15]. Hyaluronic acid (HA) is an anionic glycosaminoglycan, which can be found in the extracellular matrix [16]. HA shows great features such as biosafety, biodegradability and non-immunogenicity, which make it a good candidate for drug encapsulation [17]. Furthermore, HA can recognize and attach to the cell surface CD44 receptor, which is often upregulated in many cancer cells and represents the aggressiveness of breast tumor cells [18, 19].

Melittin (Mel) is a 26-amino acid peptide obtained from European bee venom [20]. It has been demonstrated that this peptide has antitumor activity towards several malignancies through induction of tumor necrosis or apoptosis by disrupting cell membrane integrity together with activating caspase and matrix metalloproteinase [21, 22]. In addition, this cationic peptide is able to act as an immunostimulatory adjuvant to stimulate immune system against tumor cells [23]. Therefore, it is an excellent candidate to be employed as both a chemotherapeutic and immunotherapeutic agent.

In this paper, we introduce a simplified approach for targeted delivery of various therapeutic agents to cancer cells to achieve efficient cancer chemo- and immunotherapy. Mel was bound to PD-L1 DsiRNA (Dicer-substrate short-interfering RNA) through electrostatic interaction to combine the synergetic effect of chemotherapy and immunotherapy. Then, doxorubicin (Dox), a well-known chemotherapeutic drug, was loaded onto the developed nanocomposite to improve the efficiency of the proposed treatment. In order to enhance the biocompatibility, safety, stability, and specificity of the designed nanocomplex, the surface of the developed nanocomposite was modified with HA (Scheme 1). Not only does HA increase the accumulation of prepared complex in tumor region owing to the targeting ability of HA via binding to CD44, but also hyaluronidase in the extracellular matrix can assist in the degradation of HA, resulting in the release of therapeutic modalities in the tumor microenvironment. After evaluation of the physicochemical properties of the prepared nanoparticles, including size, surface charge, and shape, the safety and efficiency of the DoxMel/PD-L1 DsiRNA@HA were intensely assessed through various in vitro and in vivo experiments.

Cell Culture

The 4T1 cells (CRL-2539, mouse triple-negative mammary tumor cell line isolated from Balb/c mice), MDA-MB-231 cells (HTB-26™, human triple-negative mammary tumor cell line) and RAW 264.7 cells (TIB-71, macrophage-like cell line derived from Balb/c mice) were obtained from American Type Culture Collection (ATCC). 4T1 cells and RAW 264.7 cells were cultured in Roswell Park Memorial Institute Medium (RPMI) and MDA-MB-231 cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and a penicillin/streptomycin cocktail (100 µg/mL).

Construction Of Mel/pd-l1 Dsirna

Briefly, 100 µl DsiRNA (Integrated DNA Technologies) at a final concentration of 10 µM was added to 100 µl Mel (> 85% in purity, M2272, Sigma-Aldrich) at different Mel/DsiRNA ratios (0.5:1, 1:1, 2:1, 3:1 and 4:1). The mixture was then incubated for 1 h in room temperature, followed by electrophoresis using 2.5% agarose gel to assess the formation of Mel/PD-L1 DsiRNA.

Stability Of Mel/pd-l1 Dsirna

The stability of Mel/PD-L1 DsiRNA was evaluated in the presence of RNase. In brief, RNase (R1253, ThermoFisher) was added into solutions containing either PD-L1 DsiRNA or Mel/PD-L1 DsiRNA. After 2-hour incubation at room temperature, the structure of PD-L1 DsiRNA and Mel/PD-L1 DsiRNA was evaluated by applying 2.5% agarose gel electrophoresis.

Preparation Of Doxmel/pd-l1 Dsirna

Twenty microlitre of Dox (15007, Cayman Chemical) at a final concentration of 20 µM was added to the solution with increasing concentrations of Mel/PD-L1 DsiRNA. Following incubation at room temperature for 2 hours, the amount of Dox loaded onto Mel/PD-L1 DsiRNA was determined through measuring the fluorescence of Dox at the excitation of 480 nm using SpectraMax iD3 microplate reader.

Preparation Of Of Doxmel/pd-l1 Dsirna@ha

To generate DoxMel/PD-L1 DsiRNA@HA, 300 µl HA (ab143634, Abcam) at a concentration of 10 mg/ml was added to 1 ml of DoxMel/PD-L1 DsiRNA solution and stirred for 3 hours. After that the final product (DoxMel/PD-L1 DsiRNA@HA) was collected by centrifugal device. The size and zeta potential of collected particle and other intermediate products were assessed by dynamic light scattering (DLS, Malvern Zetasizer). Moreover, transmission electron microscopy (TEM, HT-7700, Hitachi, Japan) was applied to observe the morphology of DoxMel/PD-L1 DsiRNA@HA.

Drug Release Study

The effect of pH on Dox release was measured using centrifugal devices. Briefly, DoxMel/PD-L1 DsiRNA@HA was immersed into phosphate buffer saline and sodium acetate buffer (pH 7.4 or 5.5, respectively). At specific time points (1, 3, 6, 12, 24 and 36 hours), DoxMel/PD-L1 DsiRNA@HA was collected from the buffer by 3K centrifugal device and the fluorescent intensity of buffer, which represents the amount of the released Dox, was measured using SpectraMax iD3 microplate reader. The accumulative release percentage (AR %) of Dox was calculated by applying the equation of AR % = D₁/D₀ × 100, where D₁ is the concentration of released Dox and D₀ is the concentration of Dox loaded onto DoxMel/PD-L1 DsiRNA@HA.

Protein Expression Of Cd44 In Tnbc Cells

To measure the expression level of CD44, RAW264.7, 4T1 and MDA-MB-231 cells were subjected to western blot analysis using CD44 antibody (15675-1-AP, Proteintech).

Measurement Of Pd-l1 Expression

The 4T1 cells were cultured in 24-well plates and treated with Mel/PD-L1 DsiRNA and Mel/PD-L1 DsiRNA@HA (final concentration of siRNA for both treatments was 200 nM). After incubation for 48 hours, cells were lysed in buffer (10 mm HEPES pH 7.4, 50 mm Na pyrophosphate, 50 mm NaF, 50 mm NaCl, 5 mm EDTA, 5 mm EGTA, 100 µm Na3VO4, and 0.1% Triton X-100) and the expression of PD-L1 was evaluated by western blotting using PD-L1 antibody (66248-1-Ig- Proteintech).

In vitro cytotoxicity assay

The 4T1 cells were seeded onto 96-well plates with 5 × 10³ cells per well (n = 5). The next day, cells were treated with Dox, Mel, DoxMel/PD-L1 DsiRNA and DoxMel/PD-L1 DsiRNA@HA. The concentrations of Dox and Mel were 1 and 2 µM in each treatment, respectively. After 24 and 48 hours of treatment, cells were incubated with 10 µl of MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) solution (G9243, Promega) for 3 hours. Finally, a microplate reader (BioTek Synergy H1) was employed to measure absorbance of each sample at 490 nm. The optical density (OD) values of sham cells were set at 100% viability, and the percentage of viability of treated cells was calculated accordingly.

Apoptosis Analysis

To evaluate the impact of the developed treatment on apoptosis, 4T1 cells were seeded onto 12-well plates for 24 hours, followed by incubation with Dox, Mel, DoxMel/PD-L1 DsiRNA and DoxMel/PD-L1 DsiRNA@HA. The concentrations of Dox and Mel were 1 and 2 µM in each treatment, respectively. After 24 hours, cells were collected and resuspended in annexin binding buffer (V13246, Thermofisher Scientific). Subsequently, 5 µl of the annexin V- Fluorescein isothiocyanate (FITC) staining (A13199, Thermofisher Scientific) reagent was added to the cells, followed by incubation in dark for 20 minutes. Finally, flow cytometry was employed to analyze the stained cells. Results were analyzed with FlowJo version 10 software.

Induction Of Immunogenic Cell Death (Icd)

The exposure of cellular calreticulin was examined to assess the effect of the designed treatment on ICD. Briefly, 4T1 cells were seeded onto a 12-well plate and treated with Dox, Mel, DoxMel/PD-L1 DsiRNA and DoxMel/PD-L1 DsiRNA@HA. The concentrations of Dox and Mel were 1 and 2 µM in each treatment, respectively. After 24 hours, cells were collected and incubated with calreticulin (Alexa Fluor® 647) antibodies (ab196159, Abcam) at 4°C in the dark for 1 hour. Then cells were washed and subjected to flow cytometry analysis.

The amount of extracellular high mobility group box 1 (HMGB1) protein after each treatment was evaluated by western blotting. In brief, supernatant of cells after treatment was collected. An equal volume of methanol and 0.25 volume of chloroform was added to each supernatant. After mixing them thoroughly, samples were centrifuged for 10 mins at 20,000 ⋅ g. The upper phase was discarded and 500 µl methanol was added into the interphase, followed by another round of centrifugation for 10 mins at 20,000 × g. At the end, protein pellet was dried at 55^oC, resuspended in protein loading buffer and western blotting was performed for evaluation of HMGB1.

Furthermore, RealTime-Glo™ extracellular ATP assay kit (GA5010, Promega) was applied to measure the level of released ATP into the supernatant. For this aim, after incubation of cells with each treatment for 36 hours, 20 µl of 4× RealTime-Glo™ extracellular ATP assay reagent was added into each well, and luminescence was measured employing a microplate reader (BioTek Synergy H1).

Animal Studies

Female Balb/c mice (aged 6–8 weeks, 000651, The Jackson Laboratory) were used for animal studies. All animal experiments described below were performed under the guidance and approval of Animal Care Committee at the University of British Columbia (A18-0275).

In vivo anti-tumor performance

The 4T1 cells (5 × 10⁵ cells) were subcutaneously implanted into the right flank of mice to establish tumors. After tumors reached ~ 50 mm³, mice were randomly separated into 5 groups: 1- mice received PBS as control, 2- mice received Dox, 3- mice exposure to Mel, 4- mice received DoxMel/PD-L1 DsiRNA, 5- mice treated with DoxMel/PD-L1 DsiRNA@HA. All mice received intratumoral injection twice on day 0 and 4. The concentration of Dox, Mel and PD-L1 DsiRNA was the same for all groups and detailed as follows: 2 mg/kg Dox, 4 mg/kg Mel, and 1 mg/kg PD-L1 DsiRNA. Following treatment, tumor size was measured using a digital caliper and calculatedthrough the following equation: (volume = length × width² × 0.52). Tumor suppression rate (TSR) was measured as TSR (%) = [1 − (tumor volume of the treated group)/(tumor volume of the control group)] × 100 (%). Mice were euthanized once they showed any of the following signs according to our protocol: ≥ 20% of their initial body weight loss, ulceration observed in ≥ 10% of tumor region, ≥ 1.7 cm of tumor size in diameter or ≥ 10% of tumor weight over body weight. Furthermore, to investigate the efficacy of each treatment, the survival rate of each group was documented. At the end of the experiment, tumor tissues were collected to examine the rate of apoptosis using Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay based on the manufacturer’s protocol (G3250, Promega).

Safety Evaluation Of In Vivo Treatments

To evaluate the safety profile of each designed treatment, body weight of mice was recorded every three days until the end of the experiment. Moreover, at 15 days post treatment, a different group of mice (n = 4) was euthanized and main tissues were collected for the assessment of potential drug toxicities by hematoxylin-eosin (H&E) staining.

Analysis Of Pd-l1 Expression And Immune Response

The expression of PD-L1 in tumor tissues was assessed through immunofluorescence staining. In brief, mouse 4T1 tumors were collected at 15 days post-treatment, fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 and blocked with 10% FBS. Tumors were then subjected to staining with PD-L1 antibody overnight, followed by incubation with Alexa Fluor® 488-conjugated secondary antibody (A11029, Invitrogen) for 1 hour. Finally, tumors were mounted with DAPI and the slides were scanned under Zeiss LSM 880 inverted confocal microscopy. For analysis of infiltration of immune cells into tumor microenvironment, tumor sections were deparaffinized, rehydrated, and then stained with anti-CD8 (sc-1177, Santa Cruz Biotechnology), anti-NK1.1 (14-5941-82C, eBioscience), and anti-F4/80 (sc-377009, Santa Cruz Biotechnology) antibodies. Immunohistochemistry (IHC) was performed using the MACH4 Universal HRP-Polymer Detection System (BRI4012H, Biocare Medical) and hematoxylin solution Gill II (GHS232, Sigma-Aldrich) as previously noted [24], Lastly, Aperio ScanScope AT (Digital slide scanner, Leica Biosystems Inc) was employed to obtain whole-slide digital images. Quantification analysis was conducted using NIH ImageJ (version 1.52p) and presented as relative optical density. In addition, the level of proinflamatory cytokines, including IFN-γ, IL-6 and TNF-α, as well as granzyme B in tumor microenvironment was evaluated via immunofluorescence staining using anti-IFN-γ (505802, Biolegend), anti-IL-6 (504502, Biolegend), anti-TNF-α (506302, Biolegend), and granzyme B antibody (4275, Cell signaling).

Distant Tumor Study

A bilateral tumor model was established to assess the impact on distant tumors. Briefly, 4T1 cells (2 × 10⁵ cells) were subcutaneously injected into the right flank of mice. Three days later, mice were rechallenged with another subcutaneous injection of 4T1 cells (2 × 10⁵ cells) into the left flank. Once primary tumors on the right flank reached ~ 80mm³, mice were received intratumoral (right flank) injection of different formulations (as above). The length and width of the distant tumors were monitored every three days using a digital caliper.

Formation of Mel/PD-L1 DsiRNA and stability evaluation

Gel retardation procedure was employed to investigate the optimal concentration of Mel that can absorb a certain amount of DsiRNAs. As shown in Fig. 1A, at the Mel/DsiRNA ratio of 3:1, no free DsiRNA band was observed, suggesting that all DsiRNA binds to Mel. Therefore, this ratio was considered as the optimal concentration of Mel to absorb the entire dose of DsiRNA used for this study. The stability of the prepared complex was evaluated by 2.5% agarose gel in the presence of nuclease. The results demonstrated that the free form of DsiRNA disappeared after introduction of nuclease into the solution, while the DsiRNA within the Mel/DsiRNA complex shown at the top of the gel was unaffected (Fig. 1B), indicating that Mel can protect DsiRNA from degradation.

Loading Of Dox Onto Mel-dsirna Structure

Fluorometric analysis was performed to verify proper formation of DoxMel/PD-L1 DsiRNA complex by measuring the fluorescence of Dox. Intercalation of Dox with PD-L1 DsiRNA in Mel/PD-L1 DsiRNA complex leads to quenching of fluorescence intensity of Dox [25]. Fluorescence signal of fixed concentration of Dox (20 µM) was evaluated after being added to different concentrations of Mel/PD-L1 DsiRNA. As displayed in Fig. 1C, the maximal quenching of the fluorescence intensity of Dox was obtained in the Mel/PD-L1 DsiRNA with 10 µM of DsiRNA. Therefore 2:1 for Dox and DsiRNA was regarded as the optimal ratio and employed for subsequent experiments.

Synthesis And Characterization Of Doxmel/pd-l1 Dsirna@ha

DoxMel/PD-L1 DsiRNA@HA was prepared through electrostatic interaction between negative-charged HA and positive-charged DoxMel/PD-L1 DsiRNA. DLS was employed to monitor formation of DoxMel/PD-L1 DsiRNA@HA at each step. Figure 1D showed that DoxMel/PD-L1 DsiRNA had a zeta potential of 7.38, while its modification with HA decreased the zeta potential to -11.9. Moreover, the average size of DoxMel/PD-L1 DsiRNA and DoxMel/PD-L1 DsiRNA@HA was approximately 134 ± 32.79 and 219 ± 32.16 nm, respectively (Fig. 1E). These alterations in zeta potential and size verify the proper encapsulation of DoxMel/PD-L1 DsiRNA into HA.. Finally, TEM revealed that DoxMel/PD-L1 DsiRNA@HA exhibited a spherical shape with the size of ~ 250 nm (Fig. 1F), consistent with DLS results, confirming the successful preparation of DoxMel/PD-L1 DsiRNA@HA.

Drug Release

To investigate the release behavior of Dox from DoxMel/PD-L1 DsiRNA@HA nanoparticles, developed complex was incubated under pH 5.5, which represents acidic environment of endosomes, and pH 7.4 of physiological conditions for 36 hours. Figure 1G showed that around 80% of the Dox was released at the acidic pH condition compared with ~ 40% of Dox release under pH 7.4 at 36 hours. This feature is considered as an advantage of developed nanoparticle as the tumor microenvironment is acidic, favorable for Dox release, and thereby preventing off-target effects caused by free drug administration.

In vitro studies

Cellular Internalization And Knockdown Efficiency

We first examined the protein levels of CD44 (cell surface receptor for HA) in various cell types. We found that both 4T1 cells and MDA-MB-231 TNBC cells expressed CD44, while CD44 expression is undetectable in mouse RAW264.7 macrophages (Fig. 2A). Flow cytometry and confocal microscopy were applied to evaluate the cellular uptake of DoxMel/PD-L1 DsiRNA@HA. We showed that encapsulation of therapeutic agents in HA enhanced uptake efficiency by approximately 3-fold compared with DoxMel/PD-L1 DsiRNA and free Dox in 4T1 cells (Fig. 2B). Similarly, confocal microscopy revealed that the fluorescent signal was significantly higher (around 2.5-fold) in 4T1 cells treated with DoxMel/PD-L1 DsiRNA@HA than cell treated withfree Dox or DoxMel/PD-L1 DsiRNA (Fig. 2B).Furthermore, we showed that HA modification significantly increased the knockdown efficiency of PD-L1 DsiRNA (Fig. 2D).

Cytotoxicity Assessment

MTS assay was performed to assess the cytotoxicity of the developed nanomedicine in 4T1 cells. Compared with single administration of Dox and Mel, combination therapy using DoxMel/PD-L1 DsiRNA resulted in around 20% lower cell viability (Fig. 3A). However, the highest cytotoxicity was detected in cells treated with DoxMel/PD-L1 DsiRNA@HA for either 24 or 48 hours (Fig. 3A). To further evaluate the cytotoxicity of different formulations, annexin V-FITC staining was conducted by flow cytometric analysis. As shown in Fig. 3B, the level of apoptotic cells was 45.76% and 55.68% after treatment of 4T1 cells with DoxMel/PD-L1 DsiRNA and DoxMel/PD-L1 DsiRNA@HA, respectively, which was about 2-fold higher compared with single treatment. Togerther, these findings imply the successful delivery of therapeutic agents to cancer cells and the great synergistic effect of agents in inducing cell death.

Induction Of Immunogenic Cell Death

There is growing evidence demonstrating that ICD is critical for antitumor immunity. Exposure of calreticulin as well as the release of HMGB1 protein and ATP into the extracellular milieu are the main markers of ICD [26]. In the next step, we assessed the induction of ICD in 4T1 cells after using different formulations. As shown in Fig. 3C, secretion of ATP into extracellular environment amplified by 4-fold after treatment of cells with Dox and Mel compared with control group. Although DoxMel/PD-L1 DsiRNA enhanced the secretion of ATP to 57.39%, the highest extracellular ATP was found in cells treated with DoxMel/PD-L1 DsiRNA@HA, in which ATP level reached 74.15% (Fig. 3C). Consistent with these findings, a significantly higher release of HMGB1 proteins was observed in cells treated with DoxMel/PD-L1 DsiRNA@HA in comparison with other formulations (Fig. 3D). Lastly, we demonstrated that DoxMel/PD-L1 DsiRNA@HA-treated cells exhibited the highest proportion of calreticulin-positive cells (54.11%), as compared to DoxMel/PD-L1 DsiRNA- (35.43%), Dox- (25.67%), and Mel-treated (24.48%) cells (Fig. 3E). Collectively, our results verify effective ICD induction by targeted combination therapy.

In vivo anti-tumor study

We next sought to determine the anti-tumor efficacy of the developed nanoparticales in vivo using the 4T1 tumor-bearing Balb/c mouse model. The experimental procedure is outlined in Fig. 4A. We found that the DoxMel/PD-L1 DsiRNA@HA complex was able to reduce tumor size the most significantly compared with other treatments (Fig. 4B). On day 21, the average tumor size for mice that received DoxMel/PD-L1 DsiRNA@HA complex was 198.92 ± 102 mm³, while mice treated with DoxMel/PD-L1 DsiRNA, Dox or Mel showed the average tumor size of 354.74 ± 30.22 mm³, 455.23 ± 68.73 mm³ and 731.95 ± 97.36 mm³, respectively, vadilating the efficiency of combination therapy in suppressing tumor growth (Fig. 4B).. Similarly, DoxMel/PD-L1 DsiRNA@HA-treated group displayed the lowest tumor weight (Fig. 4C) and the highest TSR (Fig. 4D) among all treatment groups. These findings highlight the synergetic effects of different therapeutic agents and the importance of HA in drug delivery. Additionally, survival analysis exhibited that DoxMel/PD-L1 DsiRNA@HA was able to prolong the survival of 4T1-bearing mice more significantly compared with other agents (Fig. 4E), signifying the role of HA as both a drug encapsulator and a targeting agent for efficient delivery of therapeutic agents in tumor microenvironment. Furthermore, TUNEL assay revealed that the highest apoptosis rate was observed in mice treated with DoxMel/PD-L1 DsiRNA@HA compared with non-targeted combination therapy and single treatment (Fig. 4F). Finally, the H&E staining of major tissues showed that none of the treatments caused significant toxicity (Fig. 4G). There were also no significant differences in the body weight of mice treated with different formulations compared with sham group (Fig. 4H). These outcomes suggest a good safety profile of the proposed treatment in the animal model.

Measurement Of Pd-l1 Proteins And Proinflammatory Cytokines In The Tumor Microenvironment

We further evaluated the density of PD-L1 in the tumor microenvironment using immunofluorescence staining. While no significant differences in PD-L1 expression were observed between mice in the sham group and mice received Dox and Mel, a detectable reduction in PD-L1 was found in tumors of mice treated with either DoxMel/PD-L1 DsiRNA or DoxMel/PD-L1 DsiRNA@HA compared with the sham group, with the latter being the most potent in such action (Fig. 5A). The finding implies the importance of HA in packaging and efficient delivery of components to cancer cells. Proinflammatory cytokines play a crucial role in the stimulation of immune cells. Immunofluorescence analysis revealed that proinflammatory cytokines, including IFN-γ, TNF-α and IL-6, were barely detectable in tumor sections of mice that received PBS, Dox and Mel (Fig. 5B). However, the level of pro-inflammatory cytokines increased in the mice treated with DoxMel/PD-L1 DsiRNA, while the strongest intensity signal was observed in mice that received DoxMel/PD-L1 DsiRNA@HA, further validating the efficacy of targeted combination therapy (Fig. 5B).

Infiltration Of Immune Cells Into The Tumor Microenvironment And Distant Tumor Study

After assessing PD-L1 and proinflammatory cytokine expression, we then performed immunohistochemical and immunofluorescent staining to investigate immune cell infiltration into the tumor microenvironment and granzyme B expression, respectively. We demonstrated that mice treated with DoxMel/PD-L1 DsiRNA@HA exhibited the highest level of infiltrated macrophages, NK cells, and T cells (Fig. 6A) and the greatest intensity of granzyme B signal in the tumors (Fig. 6B). These findings imply that a high accumulation of therapeutic agents in tumor regions, followed by downregulation of PD-L1 mediated by PD-L1 DsiRNA, and induction of ICD by Dox and Mel as well as the release of proinflammatory cytokines led to strong activation of the immune system, which further contributes to inhibition of tumor growth.

To further investigate the involvement of immune system in tumor suppression, a bilateral tumor model was established. Primary tumors were treated as described earlier and the growth of distant tumor was monitored. Consistent with the antitumor study above, administration of DoxMel/PD-L1 DsiRNA@HA triggered the strongest regression of distant tumors with an impressive tumor inhibitory rate (73.79%) compared with other treatments (Fig. 6C and B). This outcome suggests that the proposed targeted combination therapy is an efficient activator of the immune system to achieve robust cancer immunotherapy.

In summary, an efficient therapeutic system was designed and developed for targeted chemo-, immunotherapy. Our results indicate that encapsulation of therapeutic agents with HA significantly improves delivery efficiency. Upon reaching tumor cells, acidic condition assists in release of Dox, Mel, and PD-L1 DsiRNA. While Dox and Mel are able to kill cancer cells and induce ICD, application of PD-L1 DsiRNA significantly suppresses PD-L1 expression. Induction of ICD, together with PD-L1 knockdown facilitate strong immune cell stimulation to suppress tumor growth. As this treatment strategy was built upon binding of HA to CD44 for efficient delivery of therapeutic agents, we expect that the same approach can be applied against other types of tumors, in which CD44 is overexpressed.

Ethics approval and consent to participate

All animal experiments described below were performed under the guidance and approval of Animal Care Committee at the University of British Columbia (A18-0275).

Consent for publication

Not applicable

Availability of data and materials

All data analyzed during this study are included in this published article.

Competing interests

The authors declare that they have no competing interests.

Funding

This work was supported by the Cancer Research Society, BC Lung Association, Providence Health Care Research Institute (PHCRI) and Vancouver Coastal Health Research Institute (VCHRI) Innovation and Translational Research Award. AB is recipient of a four-year PhD Fellowship and Laurel L. Watters Research Fellowship from the University of British Columbia. YM is recipient of Michael Smith Health Research British Columbia.

Authors' contributions

AB performed the majority of the experiments and analyzed the data. AB conducted the animal studies. YM and JZ were involved to evaluate the efficacy of developed treatment in vitro. AB drafted the original manuscript. All authors participated in discussion, analyzed the results and approved the final manuscript. HLuo conceptualized the project with AB and edited the manuscript.

Acknowledgements

Not applicable

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Scheme 1 is available in Supplementary Files section.

No competing interests reported.

Scheme1.png
Scheme 1. Schematic illustration of DoxMel/PD-L1 DsiRNA@HA preparation for targeted breast cancer chemo-immunotherapy

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Engineering a facile and versatile nanoplatform to facilitate the delivery of multiple agents for targeted breast cancer chemo-immunotherapy

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Abstract

Background

Results

Conclusion

Figures

Introduction

Material And Methods

Cell Culture

Construction Of Mel/pd-l1 Dsirna

Stability Of Mel/pd-l1 Dsirna

Preparation Of Doxmel/pd-l1 Dsirna

Preparation Of Of Doxmel/pd-l1 Dsirna@ha

Drug Release Study

Protein Expression Of Cd44 In Tnbc Cells

Measurement Of Pd-l1 Expression

Apoptosis Analysis

Induction Of Immunogenic Cell Death (Icd)

Animal Studies

Safety Evaluation Of In Vivo Treatments

Analysis Of Pd-l1 Expression And Immune Response

Distant Tumor Study

Results And Discussion

Formation of Mel/PD-L1 DsiRNA and stability evaluation

Loading Of Dox Onto Mel-dsirna Structure

Synthesis And Characterization Of Doxmel/pd-l1 Dsirna@ha

Drug Release

Cellular Internalization And Knockdown Efficiency

Cytotoxicity Assessment

Induction Of Immunogenic Cell Death

Measurement Of Pd-l1 Proteins And Proinflammatory Cytokines In The Tumor Microenvironment

Infiltration Of Immune Cells Into The Tumor Microenvironment And Distant Tumor Study

Conclusion

Declarations

References

Scheme

Additional Declarations

Supplementary Files

Status:

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