Drug Delivery2018ResearchDrug Delivery

Development of dual drug loaded stealth nanocarriers for targeted and synergistic anti lung cancer efficacy

Juan Chen, Xiaobing Yang, Liuqing Huang, Huixian Lai, Chuanhai Gan, Xuetao Luo29 September 2026DOI 10.1080/10717544.2018.1477856

Summary

Lung cancer has the highest mortality among cancers, and single-agent chemotherapy is often unsuccessful due to drug resistance, insufficient intracellular uptake, nonspecific targeting, and severe systemic toxicity. Combination chemotherapy can suppress drug resistance and achieve synergistic anticancer efficacy, but there is a need for targeted nanocarrier systems that can co-deliver multiple chemotherapeutic agents while providing active. ### Nanoparticle Characterization | Parameter | Result | |---------------|------------| | Hydrodynamic diameter (DLS) | 176 nm | | TEM core diameter | ~80 nm | | Polydispersity index (PDI) | 0.109 ± 0.034 | | Zeta.

Keywords

NanocarriersCellular uptakeChitosanNanoparticlesEndocytosisDrug deliveryChemotherapy

Purpose: Lung cancer has the highest mortality among cancers, and single-agent chemotherapy is often unsuccessful due to drug resistance, insufficient intracellular uptake, nonspecific targeting, and severe systemic toxicity. Combination chemotherapy can suppress drug resistance and achieve synergistic anticancer efficacy, but there is a need for targeted nanocarrier systems that can co-deliver multiple chemotherapeutic agents while providing active targeting and sustained release.

Hypothesis: Dual-drug-loaded PEGylated chitosan nanoparticles (PCNPs) co-delivering methotrexate (MTX) and pemetrexed (PMX)—with MTX functioning both as a chemotherapeutic agent and a folate receptor-targeting ligand—will exhibit synergistic anticancer efficacy against lung cancer through enhanced cellular uptake, sustained drug release, and improved tumor accumulation via the EPR effect, resulting in superior in vitro cytotoxicity and in vivo antitumor activity compared to free drug combinations or single-drug-loaded nanoparticles.

Aims: 1. Synthesize and characterize MTX-PMX-PCNPs (PEGylated chitosan nanoparticles co-loaded with MTX and PMX) and evaluate their physicochemical properties (size, zeta potential, morphology, drug loading, and release profiles) 2. Evaluate in vitro cellular uptake of MTX-PMX-PCNPs in A549 lung cancer cells compared to free drugs and single-drug-loaded formulations 3. Assess in vitro cytotoxicity and synergism using CCK-8 assays and combination index (CI) analysis to quantify the synergistic effect of dual-drug delivery 4. Determine cell cycle effects of MTX-PMX-PCNPs in A549 cells 5. Evaluate in vivo antitumor efficacy in a Lewis lung carcinoma (LLC) mouse model by measuring tumor growth inhibition, tumor weight reduction, and histopathological changes

Delivery system:

Component: Polymer Core; Description: Chitosan (CS), 75 kDa MW, 95% deacetylation degree — biocompatible, biodegradable, mucoadhesive

Component: Stealth Coating; Description: Methoxy poly(ethylene glycol) (mPEG) — provides hydrophilic PEG shell for prolonged circulation, reduced phagocytosis, EPR effect

Component: Drug 1 (Chemotherapeutic + Targeting Ligand); Description: Methotrexate (MTX) — DHFR inhibitor; also binds folate receptors (FRs) overexpressed on tumor cells for active targeting

Component: Drug 2 (Chemotherapeutic); Description: Pemetrexed (PMX) — multi-targeted antifolate; inhibits TS, GARFT, DHFR, AICARFT

Component: Conjugation Chemistry; Description: EDC-mediated amide bond formation between carboxyl groups of MTX/PMX and primary amine groups of chitosan

Component: Drug Loading; Description: MTX LC: 22.78 ± 0.71%; PMX LC: 23.13 ± 0.55%

Component: Particle Properties (DLS); Description: • Hydrodynamic diameter: 176 nm • PDI: 0.109 ± 0.034 • Zeta potential: +47 mV

Component: Particle Properties (TEM); Description: • Core size: ~80 nm (subsphaeroidal, monodispersed)

Component: Release Profile; Description: • Sustained release over 144 h (pH-dependent) • Faster release at pH 7.4 vs. pH 6.5

Component: Targeting Mechanism; Description: MTX-mediated active targeting to folate receptors (FRs) overexpressed on tumor cells via receptor-mediated endocytosis; EPR effect for passive targeting

Approach:

Parameter: Cell Lines; Details: • A549 (human lung adenocarcinoma epithelial cells) — in vitro studies • LLC (mouse Lewis lung carcinoma) — in vivo tumor model

Parameter: In Vitro Cytotoxicity; Details: CCK-8 assay: A549 cells treated with PCNPs, free MTX, free PMX, MTX+PMX (free combination), MTX-PCNPs, PMX-PCNPs, MTX-PMX-PCNPs at concentrations 0.05-500 μg/mL for 24, 48, 72 h; IC50 values calculated

Parameter: Synergy Analysis; Details: Combination Index (CI) method (Chou-Talalay): CI < 1 = synergism, CI = 1 = summation, CI > 1 = antagonism

Parameter: Cellular Uptake; Details: Confocal laser scanning microscopy (CLSM): FITC-labeled formulations; DAPI nuclear stain; 4 h incubation

Parameter: Cell Cycle Analysis; Details: Flow cytometry (PI staining); 48 h treatment; ModFit LT software; G0/G1, S, G2/M phase distribution

Parameter: In Vivo Model; Details: C57BL/6 mice (4-week-old, 18-20 g); subcutaneous LLC tumor model (1.08 × 10⁷ cells/mouse)

Parameter: Treatment Groups; Details: • 0.9% NaCl (control) • MTX+PMX (free drug combination) • MTX-PCNPs • PMX-PCNPs • MTX-PMX-PCNPs

Parameter: Dosing; Details: 4 mg/kg (PMX-equivalent dose); tail vein injection on days 0, 3, 7, 14, 21

Parameter: Sample Size; Details: n=6 mice per group

Parameter: Efficacy Endpoints; Details: Tumor volume (measured every 5 days for 25 days); tumor weight (excised at day 25); tumor inhibition rate (TIR%); body weight monitoring

Parameter: Histopathology; Details: Hematoxylin and eosin (H&E) staining of tumor sections

Parameter: Statistical Tests; Details: One-way ANOVA with SPSS; P < 0.05 considered significant

Key methods:

Analysis Category: Nanoparticle Characterization; Methods: • FTIR (Nicolet AVATR 360) — chemical structure confirmation • TEM (JEM-2100) — morphology and core size • DLS (Marvent Nano-zs) — hydrodynamic size, PDI, zeta potential • RP-HPLC (Waters 1525, C18 column) — drug loading content (LC) and entrapment efficiency (EE)

Analysis Category: In Vitro Drug Release; Methods: Dialysis method (MWCO 3 kDa); PBS at pH 6.5 and 7.4; 37°C; 144 h; samples analyzed by RP-HPLC

Analysis Category: Cellular Uptake; Methods: CLSM (Leica TCS SP5): FITC-labeled formulations (ex/em: 488/518 nm); DAPI nuclear stain (ex/em: 364/461 nm)

Analysis Category: Cell Viability; Methods: CCK-8 assay (Dojindo); OD at 450 nm; IC50 calculation

Analysis Category: Synergy Evaluation; Methods: Combination Index (CI) method based on Chou-Talalay equation

Analysis Category: Cell Cycle Analysis; Methods: Flow cytometry (Beckman); PI staining (50 μg/mL); RNase A (100 μg/mL); ModFit LT Mac 3.3 software

Analysis Category: In Vivo Imaging/Assessment; Methods: Caliper measurements; tumor volume = length × width² / 2

Analysis Category: Histopathology; Methods: H&E staining; Axio Vert.A1 Zeiss microscope

Analysis Category: Statistical Analysis; Methods: SPSS; one-way ANOVA; P < 0.05 = significant

Key results: ### Nanoparticle Characterization

Parameter: Hydrodynamic diameter (DLS); Result: 176 nm

Parameter: TEM core diameter; Result: ~80 nm

Parameter: Polydispersity index (PDI); Result: 0.109 ± 0.034

Parameter: Zeta potential; Result: +47 mV

Parameter: MTX loading content; Result: 22.78 ± 0.71%

Parameter: PMX loading content; Result: 23.13 ± 0.55%

Parameter: MTX-PMX-PCNPs morphology; Result: Subsphaeroidal, monodispersed

In Vitro Drug Release (MTX-PMX-PCNPs, 144 h):

Condition: pH 7.4; Release Profile: Faster release vs. pH 6.5

Condition: pH 6.5; Release Profile: Slower, sustained release

Condition: Free MTX/PMX; Release Profile: ~99% released within 2 h

Condition: MTX-PMX-PCNPs (2 h); Release Profile: ~6% released

Condition: Release mechanism; Release Profile: Amide bond hydrolysis; pH-dependent

IC50 Values (A549, 72 h):

Formulation: Free MTX; IC50 (μg/mL): 4.78 ± 0.17; CI vs. free drugs: -; CI vs. single-drug NPs: -

Formulation: Free PMX; IC50 (μg/mL): 25.64 ± 0.22; CI vs. free drugs: -; CI vs. single-drug NPs: -

Formulation: MTX + PMX (free combo); IC50 (μg/mL): 8.81 ± 0.09; CI vs. free drugs: 1.12 ± 0.11 (antagonism); CI vs. single-drug NPs: -

Formulation: MTX-PCNPs; IC50 (μg/mL): 1.56 ± 0.06; CI vs. free drugs: -; CI vs. single-drug NPs: -

Formulation: PMX-PCNPs; IC50 (μg/mL): 11.65 ± 0.19; CI vs. free drugs: -; CI vs. single-drug NPs: -

Formulation: MTX-PMX-PCNPs; IC50 (μg/mL): 0.76 ± 0.04; CI vs. free drugs: 0.10 ± 0.03 (strong synergism); CI vs. single-drug NPs: 0.28 ± 0.06 (synergism)

Cell Cycle Analysis (A549, 48 h):

Treatment: Control; % in S Phase: 30.75%; Effect: Baseline

Treatment: PCNPs (empty); % in S Phase: Similar to control; Effect: No effect (carrier non-toxic)

Treatment: MTX + PMX (free); % in S Phase: 46.28%; Effect: S phase arrest

Treatment: MTX-PCNPs; % in S Phase: 39.64%; Effect: S phase arrest

Treatment: PMX-PCNPs; % in S Phase: 46.02%; Effect: S phase arrest

Treatment: MTX-PMX-PCNPs; % in S Phase: 61.93%; Effect: Most potent S phase arrest

Cellular Uptake (A549, CLSM):

Formulation: Free MTX+PMX; Fluorescence Signal: Hardly any signal (rapid excretion)

Formulation: PCNPs (empty); Fluorescence Signal: Minimal

Formulation: MTX-PCNPs; Fluorescence Signal: Stronger than free drugs

Formulation: PMX-PCNPs; Fluorescence Signal: Stronger than free drugs

Formulation: MTX-PMX-PCNPs; Fluorescence Signal: Strongest intracellular fluorescence

In Vivo Antitumor Efficacy (LLC Mouse Model, Day 25):

Group: 0.9% NaCl; Tumor Weight (g): 3.08; Tumor Inhibition Rate (TIR %): -; Body Weight Change: Stable

Group: MTX+PMX (free); Tumor Weight (g): 1.65; Tumor Inhibition Rate (TIR %): 46.43%; Body Weight Change: Decreased (toxicity)

Group: MTX-PCNPs; Tumor Weight (g): 1.35; Tumor Inhibition Rate (TIR %): 56.17%; Body Weight Change: Stable

Group: PMX-PCNPs; Tumor Weight (g): 1.56; Tumor Inhibition Rate (TIR %): 49.35%; Body Weight Change: Stable

Group: MTX-PMX-PCNPs; Tumor Weight (g): 0.58; Tumor Inhibition Rate (TIR %): 81.17%; Body Weight Change: Stable (no toxicity)

Histopathology (H&E, Day 25):

Group: 0.9% NaCl; Histological Findings: Closely packed tumor cells, intense staining, regular array

Group: MTX+PMX (free); Histological Findings: Small necrosis, reduced staining

Group: MTX-PCNPs / PMX-PCNPs; Histological Findings: Moderate necrosis, less intense staining

Group: MTX-PMX-PCNPs; Histological Findings: Lower nuclear-cytoplasmic ratio, more significant necrotic region, poorly defined borders, weak staining

Interpretation: The authors conclude that "this combination nano-delivery strategy could be considered as a prospective synergistic targeting anticancer chemotherapeutic agent, especially for carcinoma of advance lungs." The MTX-PMX-PCNPs demonstrated "a robust synergistic anticancer efficacy in lung cancer in mice" with an 81.17% tumor inhibition rate—"about 0.5 folders stronger than other groups." The authors emphasize that MTX serves a dual role as both a chemotherapeutic agent and a "lung tumor targeting ligand" via folate receptor-mediated endocytosis. The CI value of 0.10 (vs. free single drug) represents "strong synergism," while free drug combination was slightly antagonistic (CI = 1.12). The "prolonged blood circulation, more tumoral location accumulation" via stealth PEGylation and the EPR effect, combined with MTX-mediated active targeting, provides "a prospective strategy for lung cancer treatment."

10. Limitations (Explicitly Stated or Evident):

1. MTX dual role not fully distinguished: MTX functions as both a targeting ligand and a chemotherapeutic agent; the contribution of each role to the observed efficacy was not independently quantified.

2. No folate receptor expression validation: The paper assumes FR overexpression on A549 and LLC cells but does not quantify FR expression levels (e.g., by flow cytometry or Western blot) to confirm targeting suitability.

3. Limited in vivo duration: Mice were sacrificed at day 25; long-term survival benefit, tumor recurrence, or metastasis were not assessed.

4. No toxicity data beyond body weight: While body weight remained stable and behavioral observations were made, no serum biochemistry (ALT, AST, creatinine), organ histopathology, or immunotoxicity studies were performed.

5. No free MTX control for targeting: The study does not include a competitive inhibition experiment (e.g., free MTX blocking) to confirm that the enhanced uptake is due to FR-mediated endocytosis rather than other mechanisms.

6. Nano-carrier vs. free drug dose equivalence: The dosing was based on PMX-equivalent dose (4 mg/kg), but the MTX content in dual-drug nanoparticles was not matched to free MTX dose groups, potentially confounding the comparison.

7. Release study limitations: In vitro release was conducted in PBS, not in serum-containing media or in the presence of enzymes; intracellular release kinetics were not studied.

8. Cell line limited: Only A549 (human adenocarcinoma) and LLC (mouse) cells were tested; evaluation in other lung cancer subtypes (e.g., SCLC) or patient-derived cells was not performed.

9. No mechanism of synergy: The paper demonstrates synergistic efficacy but does not mechanistically explore how MTX and PMX interact at the molecular level to produce synergy (e.g., effects on folate metabolism enzymes, nucleotide pools, or signaling pathways).

10. Chitosan batch variability: Chitosan properties (MW, deacetylation degree) can vary between batches, potentially affecting reproducibility of nanoparticle synthesis.

11. No pharmacokinetic study: Blood circulation time, biodistribution, and tumor accumulation of MTX-PMX-PCNPs were not directly measured (only inferred from in vivo efficacy).

12. No comparison to other targeting ligands: The study does not benchmark MTX-mediated targeting against other FR-targeting ligands (e.g., folic acid) or non-targeted controls with equivalent drug loading.

Report prepared based on the published Drug Delivery article. For full experimental details and complete references, please refer to the original publication.

Biochemical Pharmacology*, Volume 173, 2020, Article 113648 DOI/

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