Co-delivery of etoposide and cisplatin in dual-drug loaded nanoparticles synergistically improves chemoradiotherapy in non-small cell lung cancer models

Abstract

Chemoradiotherapy with cisplatin and etoposide is a curative management regimen for both small and non-small cell lung cancers. While the treatment regimen is effective, it also has a high toxicity profile. One potential strategy to improve the therapeutic ratio of chemoradiation is to utilize nanotherapeutics. Nanoparticle formulation of cisplatin and etoposide, however, is challenging due to the significant mismatch in chemical properties of cisplatin and etoposide. Herein we report the formulation of a polymeric nanoparticle formulation of cisplatin and etoposide using a prodrug approach. We synthesized a hydrophobic platinum prodrug, which was then co-delivered with etoposide using a nanoparticle. Using mouse models of lung cancer, we demonstrated that dual-drug loaded nanoparticles are significantly more effective than small molecule chemotherapy in chemoradiotherapy. These results support further investigation of nanoparticle-based drug formulations of combination chemotherapies and the use of nanotherapeutics in chemoradiotherapy.

Statement of Significance

The treatment of lung cancer often involves a combination of chemotherapy and radiation. While it can be effective, it also has a high toxicity profile. Preferential delivery of chemotherapeutics to the tumor while avoiding normal tissue would improve efficacy and lower toxicity. While this is challenging with conventional drug delivery technologies, nanotechnology offers a unique opportunity. In this study, we have engineered nanoparticles that are loaded with combination chemotherapeutics and showed such nanotherapeutics are more effective and less toxic than free chemotherapeutics in chemoradiotherapy. Our work highlights the importance and potential of nanoformulations of combination chemotherapy in chemoradiotherapy and cancer treatment. This approach can be translated clinically and it can have a significant impact on cancer treatment.

1. Introduction

Lung cancer is the second most common type of cancer for both men and women, behind only prostate and breast cancer, respectively. But with an estimated 229,000 new cases and over 135,000 deaths in the US in 2020, lung cancer is the leading cause of cancer deaths in the US, attributing to more cancer-related deaths than breast and prostate cancer combined [1]. Two main subtypes of lung cancer include small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). One of the curative treatments for both SCLC and NSCLC is chemoradiotherapy (CRT) [2–4]. In these CRT regimens, cisplatin (CP) with etoposide (ET) drug combinations are commonly utilized with radiotherapy [5–13]. While effective, this treatment is also highly toxic [13–17]. Thus, there has been strong interest in further improving the CRT regimen.

One strategy to improve CRT is to utilize nanotherapeutics. Nanoparticles (NPs) have been shown to act as effective drug delivery systems (DDSs), improving toxicity profiles and treatment efficacy [18]. Dual-drug loaded NPs (DNPs) can offer synergistic drug delivery with improved efficacy over free drugs [19–22]. By engineering NPs to contain a payload with a controlled drug ratio, any additive or synergistic effects of the drugs can be maximized. These drug loaded NPs also result in decreased toxicity profiles and fewer side effects since a larger portion of the drug is delivered to the tumor. This occurs through preferential accumulation of NPs at the tumor due to the enhanced permeability and retention (EPR) effect [18]. Our research investigated NP formulations of CP and ET to improve the efficacy of this combined regimen while limiting off target effects when using this drug combination in conjunction with radiotherapy (XRT) against NSCLC.

The large differences in CP and ET’s aqueous/organic phase solubility and drug loading ratios make their co-delivery challenging. To overcome this solubility difference, we employed a proven method for the modification of CP with a fatty acid to make a more hydrophobic cisplatin prodrug (CPP) to allow co-loading with ET [23–27]. We chose a biocompatible poly(lactic-co-glycolic acid)–polyethylene glycol (PLGA–PEG) polymer NP as the DDS. Using multiple NSCLC cell lines, we assessed the benefit that NPs can provide CRT using a dual-drug CP plus ET combination through in vitro cytotoxicity and in vivo tumor inhibition (Fig. 1). We found that DNPs co-loaded with a precise ratio of CP plus ET improved CRT over both free drug combinations and mixed single drug loaded NPs (MNPs).

Fig. 1.

Co-delivery of CPP and ET using PLGA–PEG DNPs improves chemoradiotherapy. DNPs both preferentially accumulate at the tumor, and also deliver a precise drug ratio to tumor cells simultaneously through all NPs being loaded with the desired dual-drug ratio.

2. Materials and methods

2.1. Materials

ET was obtained from APExBIO. PEG-PLGA (3600–30,000 MW, 50:50 LA:GA) was acquired from PolySciTech. Poly-(vinyl alcohol) (6,000 MW, 80% hydrolyzed) was acquired from Polysciences, Inc. Cell culture reagents were purchased from Gibco by Life Technologies. CellTiter 96® AQueous MTS Reagent Powder was purchased from Promega. Anti-Cisplatin modified DNA Antibody was purchased from Abcam. Cleaved Caspase-3 (Asp175) Antibody and anti-rat IgG Alexafluor®555 Antibodies were purchased from Cell Signaling. Hydrogen peroxide was purchased from Fisher Chemical. TritonX100 was purchased from PerkinElmer Life and Analytical Sciences. Crystal Violet was purchased from Thermo Fisher Scientific. All other chemicals were acquired from Sigma Aldrich and used without further purification.

2.2. Cell culture

The non-small cell lung cancer cell lines A549 (ATCC® CCL-185^TM) and H460 (ATCC® HTB-177^TM) were obtained from American Type Culture Collection (ATCC) supplied by the Tissue Culture Facility at the UNC Lineberger Comprehensive Cancer Center. The 344SQ cell line was kindly provided by Professor Chad Pecot’s lab. Cells were cultured in a complete growth medium made of DMEM (A549) or RPMI-1640 (H460 & 344SQ) medium supplemented with 10% (v v⁻¹) fetal bovine serum and 1% (v v⁻¹) penicillin/streptomycin.

2.3. CPP synthesis

CPP was synthesized as previously described elsewhere [23–27]. Verification of CPP synthesis was determined by ¹H NMR (Fig. S1) and mass spectrometry using positively charged electrospray with a measured m/z of 608.1587 and a 1.3 ppm error with a [M+Na]⁺ adduct ionization [28].

2.4. Animal maintenance

6–8 week old male nude mice weighing 27–32 g were supplied by the University of North Carolina animal facility and maintained under pathogen-free conditions in the Center for Experimental Animals (an AAALAC accredited experimental animal facility). The animal use protocol (15–301.0) was approved by the University of North Carolina Institutional Animal Care and Use Committee and conformed to the Guide for the Care and Use of Laboratory Animals [29].

2.5. Synthesis of NPs

NPs were synthesized via nanoprecipitation. Drug and polymer stock solutions were prepared in advance. ET was dissolved in acetonitrile (ACN) at a concentration of 2.5 mg mL⁻¹. CPP was dissolved in acetone at a concentration of 2 mg mL⁻¹. mPEG-PLGA (M.W. mPEG 3,600 & PLGA 30,000) was dissolved in ACN at a concentration of 20 mg mL⁻¹. Drug stock solutions were combined at varying ratios as needed, with the drug solution (volume varies) then mixed with 100 μL of polymer stock solution at a 1:1 ratio. ACN was added to make a final drug-polymer solution of 500 μL. The solution was then added drop-wise into 0.5% poly-(vinyl alcohol) deionized water (2 mL) to form a NP suspension. To remove organic solvents, the suspension was evaporated for 3 h at room temperature under an air extraction arm while stirring at 600 rpm. Next, the suspension was concentrated by ultra-centrifugation using an Amicon Ultra-4 filter (MWCO-100 kDa) at 1000 g for 15 min (EMD Millipore, Billerica, MA, USA). The concentrated NP suspension was then washed with deionized water, concentrated twice, and finally suspended in PBS.

2.6. Characterization of NPs

Size and zeta potential were determined by dynamic light scattering (DLS) (Malvern Instruments Ltd, Worcestershire, UK) with a concentrated NP suspension (10 μL) added to a cuvette with deionized water (990 μL). All tests were performed in triplicate. Transmission electron microscopy (TEM) images were obtained via a JEOL JEM 1230 TEM. Prior to imaging, concentrated NP suspensions were diluted 100x with deionized water then loaded on a 400-mesh carbon film copper grid for 1 min to adsorb. The grid was negatively stained with 2% potassium phosphotungstate (pH 7.0) for 1 min. Excess stain was removed by capillary action by touching the edge of the grid with filter paper. The grid was air dried before loading into the TEM. The stability of DNP size was evaluated under varying biological conditions including deionized water, pH 7.4 PBS, pH 6.8 PBS, DMEM medium, and DMEM medium with 10% v/v normal human serum. All tests were performed in triplicate. DNP size was first measured to establish a baseline, then 2 mg of DNPs were added to 10 mL of each solution and measured immediately after mixing, then incubated at 37 °C for 24 h with aliquots removed and measured at 1.5, 6, and 24 h. While our other NP characterization studies herein relied on reported average NP size, the addition of serum included significant measurements of particles below 40 nm in scans performed without NPs but serum only. For this reason, we instead evaluated stability based on the NP distribution peak size.

2.7. Determination of drug loading in NPs

To determine drug loading, NP samples (40 μL) were mixed with ACN (200 μL), vortexed, and sonicated for 10 min to completely break down the NP and release all drug. The treated samples were loaded and measured via a Shimadzu SPD-M20A high performance liquid chromatography (HPLC) equipped with a diode array detector and a Chromolith Fast Gradient RP-18e 50 × 2mm column (EMD. Millipore, Billerica, MA, USA). Samples (10 μL) were injected into the HPLC and eluted using a binary solvent system (phase A and B: A for Water, B for ACN) at a flow rate of 0.25 mL min⁻¹. The linear gradient program was set as follows: 0 to 20 min, 0% to 100% B; 20 to 25 min, 100% B; 25 to 30 min, 50% B; 30 to 35 min, 0% B. The column and sample temperature were maintained at 30 °C and 4 °C respectively during determination. ET (retention time 7.5 min) and CPP (retention time 12.4 min) were monitored at 220 nm.

2.8. In vitro drug release

In vitro drug-release profiles of drug loaded NPs were acquired under sink condition. NP suspensions (composed of 0.5 mL concentrated NP suspension with 1 mL deionized water) were split into aliquots (50 μL) and added into Slide-A-Lyzer MINI dialysis microtubes (MWCO 20 kDa, Pierce, Rockford, IL, USA). These microtubes were then dialyzed in PBS (4 L) under gentle stirring at 37 °C. NP samples (40 μL) were collected at the indicated times and measured by HPLC as previously described. Percentage of drug released was calculated by (1 − C_t/C₀) × 100%, where C₀ and C_t represent the remaining drug concentration at time 0 and t respectively.

2.9. In vitro cytotoxicity

Cells were plated in 96-well plates (3600 cells well⁻¹) and allowed to recover overnight. Cells were then washed with PBS and treated with varying concentrations and ratios of free drugs or drug encapsulated NPs at 37 °C for 2 h. Cells were then washed with PBS 3 times and allowed to grow in complete cell culture medium for 72 h. After incubation, cell viability was analyzed by an MTS assay per vendors instructions (Promega). Absorbance was recorded at 492 nm via a microtiter plate reader (Molecular Devices Corporation, California, USA). The IC₅₀ values of different treatments were calculated by fitting the dose-dependent cell viabilities to a four-parameter logistic model using the MasterPlex 2010 software pack (MiraiBio Group, Hitachi Solutions America, Ltd.).

2.10. Clonogenic survival assay

Cells (200, 300, 400, 800, 2000, and 5000 cells well⁻¹) were seeded in 6-well plates and incubated in complete cell culture medium for 2 h for attachment. Cells were then washed with PBS and treated with either PBS, free drug, MNPs, or DNPs for 2 h (drugs were added at their IC₂₀ dose, NPs at a 1:1.8 ET:CP/CPP ratio). Cells were washed with PBS three times and complete cell culture medium was added. Cells in 6-well plates were irradiated (X-RAD 320, Precision X-ray Inc., North Branford, CT, USA) according to the number of cells seeded (1, 2, 4, 6, and 8 Gy for 300, 400, 800, 2000, and 5000 cells well⁻¹ respectively), with the 200 cells well⁻¹ plate receiving no irradiation. The irradiated cells were allowed to grow for 14 days to generate colonies. 6-well plates with colonies were washed with PBS three times, fixed and stained with a mixture of 10% formalin and 0.5% crystal violet for 1 h, gently rinsed with tap water, and then dried in normal air at room temperature. All colonies with over 50 cells were counted. Plating efficiency was calculated via the number of colonies formed divided by the number of cells seeded. Surviving fraction (SF) of different irradiation doses was calculated by plating efficiency of irradiation treatment divided by that of non-irradiation treatment. D₀ and N were acquired by fitting the calculated SF to the single-hit multitarget model [SF = 1 − (1 − e^−D/D0)^N, where D represents the irradiation dose, D₀ the average lethal dose, and N the extrapolation number] using GraphPad Prism version 6.01 for Windows (GraphPad Software, La Jolla California USA). Sensitivity enhancement ratio (SER) was calculated using D₀ of untreated divided by that of drug treated cells.

2.11. In vivo anticancer efficacy

Murine tumor models were formed by injecting 800,000 cells (0.8 × 10⁶ cells suspended in 0.2 mL of 50% RPMI-1640 medium and 50% Matrigel® v v⁻¹) into the right flank. Tumors were allowed to grow to 80–150 mm³ before initiating treatment. Mice were divided into six groups with six mice per group. All mice received 200 μL tail vein injections on day 0 with either (1) PBS, (2) mixture of free ET (1.25 mg kg⁻¹) and CP (1.16 mg kg⁻¹), (3) PBS, (4) mixture of free ET (1.25 mg kg⁻¹) and CP (1.16 mg kg⁻¹), (5) MNPs (1.25 mg kg⁻¹ ET SNP and 2.25 mg kg⁻¹ CPP SNP), or (6) DNPs (ET 1.25 mg kg⁻¹ and CPP 2.25 mg kg⁻¹). NP drug doses listed denote drug dose, not including weight of polymer in NP. CP dosing is based on low-dose CP [30,31], CPP dosing is the molecular weight ratio of CPP/CP, and ET dosing is at the optimally identified ET:CPP synergistic molar ratio of 1:1.8 as identified in previous experiments. (Mice in groups 3, 4, 5, and 6 received X-ray irradiation (5 Gy) on day 0 (3 h after injection), day 1, and day 2. Tumor length (L) and width (W) were measured, and tumor volume was calculated using: (L*W²)/2, with L ≥ W. Body weight and tumor volume were measured and recorded on day 0, day 1, and every other day after day 1. Mice were humanely euthanized using CO₂ inhalation once tumor dimensions were larger than 2 cm in any direction, if body weight loss exceeded 20%, or if the University of North Carolina core veterinarians determined significant deterioration in general health.

2.12. In vivo hematological toxicity, hepatotoxicity and nephrotoxicity assay

In vivo toxicity was determined by sacrificing two randomly selected mice from each group on day 3. Circulating blood (0.5 mL) was collected via cardiac puncture. For hematological toxicity, whole-blood (0.5 mL) was stored in a heparin pre-treated stopper covered tube at 4 °C and analyzed for white and red blood cell counts. For hepatotoxicity and nephrotoxicity, whole-blood (1 mL) was transferred to an eppendorf tube (2 mL) and centrifuged at 900 g for 10 min to separate the plasma. The whole-blood and the isolated plasma was analyzed by the Department of Pathology & Laboratory Medicine, Animal Histopathology & Laboratory Medicine Core, University of North Carolina, for blood cell counts, aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (Crea) levels.

2.13. Immunofluorescent imaging

Tumors were excised from one euthanized mouse in each arm on day 3 after initial treatment. The tissue was washed with PBS, fixed in formalin (10% v v⁻¹), dehydrated, and then embedded in paraffin. Blank paraffin slides were hydrated and then microwaved twice for 10 min in sodium citrate solution (0.01 μm, pH 6.0). The slides were cooled to room temperature and washed with PBS. The slides were then treated with a PBS solution containing hydrogen peroxide (3% v v⁻¹) and Triton X-100 (0.5% w v⁻¹) and subsequently washed three times with PBS.

Tumor sections were mounted and then blocked with bovine serum albumin (BSA, 10% w v⁻¹) for 1 h. The slides were treated with 1:400 PBS diluted anti-cisplatin modified DNA antibody (Abcam, ab103261) or cleaved caspase-3 (Asp175) antibody (Cell Signaling, 9661S) overnight at 4 °C. Secondary antibody was then added with Anti-rat IgG (H+L) (Alexa Fluor® 555 Conjugate, Cell Signaling, 4417S) for anti-cisplatin modified DNA antibody or F(ab’)2-goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody (Alexa Fluor 594, Thermo Fisher Scientific, A11072) for anti-cleaved caspase-3 (Asp175) antibody. After 1 h, slides were washed with PBS (0.2% v v⁻¹, Tween 20) and sections were treated with DAPI (1 μg mL⁻¹, Thermo Fisher Scientific, D1306) for 20 min. Slides were washed with PBS and then sealed with fluoromount^™ aqueous mounting medium (Sigma-Aldrich, F4680–25ML) and a cover glass. Immunofluorescence images were obtained via a laser scanning confocal microscope (ZEISS, LSM 700).

2.14. Statistical methods

All experiments were performed at least three times and expressed as mean ± SD for in vitro (n = 3) or mean ± SEM for in vivo (n = 6) studies. Statistical significance was determined using two-tailed Student’s t-test except in the clonogenic study where the SERs were compared using a one-way analysis of variance (ANOVA) followed by a Tukey test and the in vivo efficacy study in which a single-tailed Student’s t-test was employed. Differences were considered significant when P < .05. All statistical analyses were completed in GraphPad Prism version 6.01 for Windows.

3. Results

3.1. Drug loading and release kinetics in vitro

Nanoprecipitation was used to formulate PEG-PLGA NPs loaded with ET and/or CPP. ET and CPP are encapsulated inside the hydrophobic NP core during nanoprecipitation. CP will not readily load into PLGA NPs so CPP was used instead to improve encapsulation. CPP can be reduced back to CP by tumor overexpressed intracellular mercaptans such as glutathione or ascorbate [32].

We examined varying drug feed ratios (wt% of drug vs polymer) (CPP 2.5–12.5%, ET 7.5–50%) to determine the optimal NP drug loading and loading ratio between ET and CPP. Encapsulation efficiency (wt% of drug encapsulated in NP vs initial feed amount) and drug loading wt% (wt% of drug vs total NP) were characterized for each formulation, first in SNPs, then in DNPs (Fig. 2, Table S1, and Table S2). In SNPs, CPP loading increased with feed ratio, reaching a maximum drug loading of 7.1 ± 0.2% at a feed ratio of 12.5%. ET SNP drug loading followed a similar trend, increasing with feed ratio and reaching a maximum drug loading of 6.8 ± 0.2% at a feed ratio of 50%. ET feed ratio was held at 25% for all further DNP studies to obtain significant ET drug loading. CPP and ET were then co-loaded to form DNPs with the CPP feed ratio ranging from 2.5–12.5%. The resulting DNP loadings illustrated in Fig. 2c and d allowed us to synthesize DNPs with ET:CPP molar ratios from 2.5:1 to 1:5. These formulations were used for in vitro efficacy studies to identify the most effective formulation.

Fig. 2.

Encapsulation efficiency (EE) and drug loading wt% (DL) of a) ET SNP, b) CPP SNP, c) ET in DNPs, and d) CPP in DNPs. a) ET encapsulation efficiency decreased, but total drug loading increased as the feed ratio was increased. b) While CPP drug loading increased with feed ratio, encapsulation efficiency remained consistent throughout. In c) and d) the ET feed ratio was kept constant at 25% in DNPs and the CPP feed ratio was varied. A CPP feed ratio of 7.5% produced the loaded 1:1.8 ET:CPP molar ratios used in all further studies in vivo.

NP size, polydispersity index (PDI), and zeta potential were also measured at each feed ratio by TEM and DLS (Fig. S2, Fig. S3, Table S3, and Table S4). ET SNPs were smaller (~70 nm) than those of CPP SNPs (~100 nm) and DNPs (~100 nm) at all feed ratios. All ET SNPs maintained a PDI below 0.1, while the PDI of CPP SNPs and DNPs increased with CPP feed ratios of 7.5% or greater. Final formulations kept the CPP feed ratio below 7.5% to keep particle size uniform for all NPs. DLS sizes were verified with TEM analysis of particle morphology, which revealed approximately spherical particles ranging from 60 to 150 nm (Fig. S3). Zeta potentials of all three NP formulations were measured, ranging from −15 to −35 mV.

Stability of DNPs was measured in varying biological media through sequential NP size measurements (Fig. S4). DNP size varied minimally after synthesis (~70nm) through 24 h of incubation with deionized water, normal PBS at pH 7.4, acidic PBS at pH 6.8, DMEM with no serum added, or DMEM with 10% v/v serum added. These studies showed that DNPs’ size was stable for 24 h in these conditions. This data is consistent with previous reports [33].

Drug release kinetics of SNPs and DNPs were evaluated under physiologic sink conditions (Fig. S5). ET released more quickly than CPP in all NP formulations, and in DNPs both drugs released more slowly than their SNP counterparts (CPP SNP t_1/2 = 7.2 h vs DNP t_1/2 = 11.1 h, ET SNP t_1/2 = 2.4 h vs DNP t_1/2 = 3.1 h).

3.2. In vitro cytotoxicity

Three NSCLC cell lines: A549, H460, and 344SQ, were used for in vitro studies. IC₅₀ values of cell lines treated with single or dual free drugs, SNPs, MNPs, or DNPs are presented in Fig. 3a and Table S5. In all cell lines, CPP and ET loaded NPs were more potent than their free drug counterparts (Fig. 3a). The IC₅₀ drug concentration of each cell line for CPP SNPs were 0.86, 0.33, and 0.31 μm respectively, and the ET SNPs were 78, 5.5, and 6.0 μm respectively. Due to the 13-fold higher ET SNP IC₅₀ concentration for A549, this cell line was excluded from all subsequent in vitro and in vivo work as this concentration level is not feasible for in vivo studies.

Fig. 3.

a) In vitro cytotoxicity of varying ET:CPP formulations including individual free drugs, dual free drugs, MNPs, and DNPs showing the most significant improvement for NPs at an ET:CPP ratio of 1:1.8 in H460 cells. b) Clonogenic assay of PBS only treatment, dual free drugs, MNPs, and DNPs resulting in modestly improved SERs for MNPs and DNPs versus dual free drugs in the 344SQ cell lines, and significantly improved SERs in the H460 cell line. No significant difference in SER was determined between MNPs and DNPs. Cells were treated with the IC₂₀ dose of their respective drug combination 2 h prior to radiation treatment with all dual-drug treatments at the ET:CPP ratio of 1:1.8. (*P < 0.05, **P < 0.01, ***P < 0.001, ns = not significant).

For each cell line, we tested multiple mixed drug ratios in addition to the individual free drugs. Free drug ET:CP ratios were varied from 4:1 to 1:4 in each cell line, and NP formulations were tested both as MNPs and DNPs with ET:CPP ratios ranging from 2.5:1 to 1:4.5. IC₅₀ values of all DNP drug ratios in the A549 and 344SQ cell lines were slightly larger than CPP SNPs (1.4- to 1.7-fold for A549, 1.3- to 1.9-fold for 344SQ), but significantly lower than ET SNPs (51- to 65-fold for A549, 10- to 15-fold for 344SQ). Differences in IC₅₀ values between DNPs and MNPs at each drug ratio in the H460 and 344SQ cell lines were not significant. While there was no significant difference between the 1:1.8 ET:CPP loaded DNP and any other DNP group in the 344SQ cell line, the IC50 level minimally decreased as CPP loading increased, suggesting additive effects in this cell line. The lowest IC₅₀ from any group was the DNP with an ET:CPP ratio of 1:1.8 in the H460 cell line. This was significantly lower than the IC₅₀ of CPP SNPs and ET SNPs (2- and 32-fold IC₅₀ improvements, with P < .01 and P < .001 respectively) but was not significantly lower than its 1:1.8 MNP counterpart. Additionally, the combination indexes of these DNPs in H460 and 344SQ cell lines were found to be 0.44 and 1.06 at ED₅₀ respectively, suggesting synergistic or at least additive effects of DNPs at this ratio (Table S6) [34]. Due to these synergistic effects and the significantly enhanced cytotoxicity as evidenced by IC₅₀ values at the 1:1.8 ET:CPP ratio, we chose to use this DNP drug ratio for all further experiments.

Typical first line therapy for locally advanced NSCLC consists of regular doses of 1.4 mg kg⁻¹ of both CP and ET in an average 70 kg, 1.9 m² person [9–11,35,36]. Our treatments were purposefully kept below this level in order to better monitor the benefits of one treatment arm over another. We used a single treatment of 1.25 mg kg⁻¹ ET and 1.16 mg kg⁻¹ CP as this was the optimal ratio we found in our in vitro work.

To examine the effects of these NPs with radiotherapy, they were studied using clonogenic survival assays (Fig. 3b). The sensitivity enhancement ratio (SER), a measure of radiosensitizer potency, was calculated for all treatment arms in the H460 and 344SQ cell lines (Fig. S9). These cell lines were each treated with dual free drugs or NP formulations and then exposed to increasing doses of radiation. IC₂₀ drug levels were used in favor of higher IC levels to prevent complete cell death when coupled with radiotherapy, allowing increased detection of discernible differences between treatment arms. In the 344SQ cell line, the free drug SER was similar to PBS. The SER of both MNPs and DNPs was much better than PBS (1.6 ± 0.1 and 1.6 ± 0.2 vs 1.0 ± 0.3) (P < .01), but only modestly better than free drugs (1.1 ± 0.5) (P = .047 and P = .055) In the H460 cell line the free drug SER compared to PBS was significantly improved (1.3 ± 0.1 versus 1.0 ± 0.1) (P < .05), as was the SER of MNPs and DNPs (1.57 ± 0.07 and 1.65 ± 0.07) compared to both PBS and free drugs (P < .05 MNP vs free drug and P < .01 DNP vs free drug). While neither cell line showed a significant difference in SER between MNPs and DNPs, these values indicate radiosensitization from free drugs in the H460 cell line and NP formulations in both cell lines.

3.3. In vivo efficacy

We then evaluated the efficacy of our NP DDS in combination with XRT in murine models using both the H460 xenograft and 344SQ allograft. Mice were either in a PBS control group or one of five treatment arms including dual free drugs, PBS+XRT, dual free drugs+XRT, MNPs+XRT, or DNPs+XRT. Drug dose, drug ratio (ET:CP/CPP = 1:1.8), and fractionated irradiation doses (5 Gy × 3d) were kept the same in applicable treatments. As shown in the tumor growth curves (Fig. 4), all drug loaded NPs significantly reduced tumor growth in both cell lines when compared to PBS only. The MNPs also significantly reduced tumor growth compared to free drugs with XRT in the 344SQ model (P < .05), but not in the H460 model. DNPs provided even greater reductions in tumor growth, significantly so when compared to free drugs in both the H460 and 344SQ models (P < .05 and P < .01). When compared to MNPs, the DNPs again provided significant tumor growth reductions in both cell lines (P < .05 in both).

Fig. 4.

In vivo efficacy of NPs in a) 344SQ and b) H460 models showing significant tumor growth inhibition from MNPs versus dual free drugs in the 344SQ model, and from DNPs versus both MNPs and dual free drugs in both models. Mice were treated once tumor volume reached 80–150 mm³. Data are represented as tumor volume (cm³) after treatment (*P < 0.05, **P < 0.01).

Tumor histology was analyzed for both cleaved caspase 3, a marker for early-stage apoptosis, and CP-DNA complexes, indicative of increased CP delivery (Fig. S8a, c, e, and g). Tissue sections from NP treatment arms (MNP and DNP) depicted a significant increase in CP-DNA adduct formation versus dual free drugs (Fig. S8b and f). This indicated increased delivery of drug to tumor tissue by NPs resulting in less systemic accumulation, and improved localization of CP to the nucleus when using NP delivery vehicles. Increases in the intensity of the apoptosis marker cleaved caspase 3 reflect the enhanced killing of tumor cells by XRT combined with our NP regimen (Fig. S8d and h).

While NPs can increase delivery to tumors by way of the EPR effect, there will also be significant distribution to clearance organs such as the liver and kidneys [37–41]. We examined if our nanoformulations caused significant toxicity to those off-target organs. Despite the potent efficacy in vivo of our DNP+XRT regimen, nephrotoxicity and hepatotoxicity in our PLGA-PEG formulated drug combinations were found to be low based on liver and kidney function indicators including aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (Crea) (Table S7 and S8). Additionally, the WBC of NP groups remained within the range of other IV treatment groups. While there was some loss of body weight following XRT, animals recovered to normal levels within approximately one week (Fig. S6 and S7).

4. Discussion

Combination CRT with CP plus ET is one of the primary treatment methods for SCLC and unresectable locally advanced NSCLC due to its superior efficacy over alternative regimens such as carboplatin with paclitaxel, despite CP plus ET’s higher toxicity. As this combination has been previously shown to be synergistic [42], we developed a biocompatible PLGA-PEG delivery vehicle which could be loaded at varying drug ratios to improve the efficacy of CRT with CP plus ET. All of our NP formulations showed improved dose response curves and IC₅₀ values in vitro over free drugs with no significant difference in IC₅₀ values between MNPs and DNPs implying consistent cellular uptake of all NPs. DNPs, however, showed significantly greater efficacy in vivo than MNPs and free drugs, indicating that only DNPs were able to continue to deliver both drugs at the precise synergistic ratio. This results from varying systemic distribution of each free drug or individually loaded NP in the MNP combination, causing a matching variation in drug ratio in tissues and tumor due to differences in characteristics such as size and surface properties (Fig. S2, Table S3). By maintaining a precise ratio of drugs that are delivered simultaneously through co-loading, our DNPs allow both drugs to act synergistically with no change in drug mechanism. While this type of synergy has been demonstrated in other studies with alternate drug combinations [19–22], our study demonstrated in vivo efficacy with a single treatment administration when others have required up to three sequential administrations. Moreover, this regimen also allowed lower CPP dosing when coupled with ET. Alternative future approaches for dual drug delivery could include incorporation of drugs loaded into gold microplates as these have been shown to offer significant drug loading [43].

In addition to improving efficacy through controlled loading ratios, low systemic toxicity was observed, similar to other studies where the observed tissue toxicity of PLGA based NPs in brain, heart, spleen, thymus, liver, kidney, and lung tissue has been shown to be no greater than typical incidental findings and that PLGA NPs are biodegradable non-toxic particles [44,45]. NPs have also been shown to reduce hepatotoxicity of NP delivered drugs due to macrophage uptake of NPs [46].

There have been significant recent advances in nanomedicine approaches including organic/inorganic hybrid NPs, artificial intelligence/machine learning to model nanomedicine, and dual drug delivery through NPs [43,47,48]. The success of precisely delivered ratios of dual drugs through NPs has also been seen in the clinic through the liposomal formulation of daunorubicin and cytarabine in VYXEOS, where a fixed 1:5 molar ratio provided synergistic treatment benefits/improved survival in acute myeloid leukemia [49]. While there have been a number of nanotherapeutics approved by the FDA for clinical use, this liposomal formulation is the only dual-drug loaded nanotherapeutic to date and the only nanotherapeutic that has shown improved efficacy/survival over its small molecule counterpart in the clinical setting [50,51]. Additionally, as acute myeloid leukemia is a cancer of the blood and bone marrow, VYXEOS does not benefit from the EPR effect. Clinical data from VYXEOS suggest that dual drug delivery can be a key strategy to improve therapeutic efficacy of nanotherapeutics. It is important to note that many other groups, including ourselves, have reported dual-drug NPs and have shown these agents are highly effective in the preclinical setting. However, there has been a lack of studies examining the use of dual-drug NPs in the chemoradiotherapy setting. Our report on NP ET/CPP in chemoradiation fills this gap and further enhances the clinical translation potential of dual-drug nanotherapeutics. As seen in our results, specific drug ratio at the tumor cells is critical to obtain synergy [21,22].

5. Conclusion

Chemoradiotherapy is a key treatment for lung cancer. Our study has demonstrated a novel strategy to further improve the therapeutic ratio of CRT for lung cancer. By engineering a nanotherapeutic that contains both CPP and ET, we increased the therapeutic efficacy of CRT in two murine lung cancer models. Further-more, we showed the treatment did not induce additional toxicity when compared to a standard CRT regimen with small molecule drugs. Our DNP highlights the potential of dual drug delivery in nanomedicine.

Abbreviations:

ACN

acetonitrile

AST

aspartate aminotransferase

BUN

blood urea nitrogen

CP

cisplatin

CPP

cisplatin prodrug

Crea

creatinine

CRT

chemoradiotherapy

DDS

drug delivery system

DL

drug loading

DLS

dynamic light scattering

DNP

dual-drug loaded nanoparticle

EE

encapsulation efficiency

EPR

enhanced permeability and retention

ET

etoposide

FR

feed ratio

MNP

mixed single drug loaded nanoparticle

NP

nanoparticle

NSCLC

non-small cell lung cancer

PDI

polydispersity index

PLGA-PEG

poly(lactic-co-glycolic acid)-polyethylene glycol

SCLC

small cell lung cancer

SER

sensitivity enhancement ratio

SNP

single drug loaded nanoparticle

TEM

transmission electron microscopy

XRT

radiotherapy