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Scientists synthesize amphiphilic polypeptides via post-polymerization modification of poly(α,L-glutamic acid) to create effective nanoparticles for the targeted delivery of hydrophobic drugs like paclitaxel, enhancing anti-cancer efficacy while minimizing toxicity to normal cells.
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- Research Question: The study investigates the potential of amphiphilic copolymers, derived from the post-polymerization modification of poly(α,L-glutamic acid), as effective delivery systems for hydrophobic drugs, specifically targeting their efficacy in loading and delivering paclitaxel.
- Research Design and Strategy: The researchers synthesized and characterized various amphiphilic copolymers by modifying poly(α,L-glutamic acid) with hydrophobic and basic amino acids as well as D-glucosamine. They aimed to evaluate the physicochemical properties of these copolymers and their ability to form nanoparticles suitable for drug delivery.
- Method: The study utilized a combination of synthesis methods including ring-opening polymerization (ROP) and post-polymerization modification. Characterization techniques such as size-exclusion chromatography (SEC), dynamic light scattering (DLS), and cell viability assays were employed to assess the properties and efficacy of the synthesized nanoparticles.
- Key Results: The synthesized nanoparticles exhibited a hydrodynamic diameter ranging from 170 to 330 nm, with effective paclitaxel loading efficiency of 80-99%. The cytostatic efficacy against human lung adenocarcinoma cells (A549) was found to be high, with IC50 values ranging from 1.3 to 7.8 ng/mL, indicating strong anti-cancer potential.
- Significance of the Research: This research contributes significantly to the field of drug delivery systems by demonstrating that amphiphilic polypeptides can effectively encapsulate hydrophobic drugs like paclitaxel, enhancing their bioavailability and therapeutic efficacy while minimizing toxicity to normal cells.
Introduction
Lung cancer remains one of the leading causes of cancer-related mortality worldwide, with non-small cell lung cancer (NSCLC) accounting for the majority of cases. The aggressive nature of this disease, characterized by late-stage diagnosis and rapid metastasis, poses significant challenges for effective treatment. Among the chemotherapeutic agents used in NSCLC, paclitaxel (PTX) is recognized for its potent anti-cancer properties. However, its clinical effectiveness is often hampered by its poor solubility and bioavailability, leading to the need for innovative strategies in drug delivery.
Traditional drug delivery methods for treating lung cancer typically involve systemic administration of chemotherapeutics, which aims to achieve therapeutic concentrations in tumor tissues. This approach often utilizes formulations such as liposomal encapsulations or solvent-based injections to enhance solubility and minimize toxicity. However, these methods face several challenges, including rapid clearance from the bloodstream, non-specific distribution, and adverse side effects on healthy tissues. Consequently, patients frequently experience significant toxicity, which can limit the dose and frequency of administration, ultimately reducing overall treatment efficacy.
The challenges associated with conventional drug delivery approaches have prompted the exploration of novel delivery systems that can enhance the solubility, stability, and targeting of chemotherapeutic agents. One innovative strategy involves the use of amphiphilic (glyco)polypeptides as drug carriers. These biocompatible and biodegradable polymers can form nanoparticles capable of encapsulating hydrophobic drugs like paclitaxel. By leveraging the unique properties of these polymers, this strategy aims to improve drug solubility, enhance targeted delivery to tumor cells, and reduce off-target effects, thereby addressing the limitations of traditional therapies and improving patient outcomes in lung cancer treatment.
Research Team and Aim
The research team was led by Dr. Evgenia G. Korzhikova-Vlakh, who conducted this study in collaboration with a multidisciplinary group of researchers including Apollinariia Yu. Dzhuzha, Irina I. Tarasenko, Leonard Ionut Atanase, and Antonina Lavrentieva. The team is affiliated with various institutions, including the Institute of Chemistry at Saint-Petersburg State University and the Russian Academy of Sciences. The study, titled “Amphiphilic Polypeptides Obtained by the Post-Polymerization Modification of Poly(Glutamic Acid) and Their Evaluation as Delivery Systems for Hydrophobic Drugs,” was published in the International Journal of Molecular Sciences.
The primary aim of the research, as articulated by Dr. Korzhikova-Vlakh, was to “synthesize and characterize amphiphilic (glyco)polypeptides, evaluate their nanoparticle formation, and assess their efficacy as delivery systems for hydrophobic drugs.” This objective highlights the team’s focus on innovating drug delivery approaches to improve the therapeutic efficacy of hydrophobic chemotherapeutics, particularly in the context of lung cancer treatment.
Experimental Process
Experiment 1: Synthesis of Amphiphilic (Glyco)Polypeptides via Post-Polymerization Modification
Primary Technique: The primary technique used was the post-polymerization modification of poly(α,L-glutamic acid) (P[E]) via activated ester chemistry, enabling the attachment of hydrophobic and basic amino acids along with D-glucosamine to create amphiphilic glycopolypeptides. This method was chosen for its simplicity and flexibility in introducing diverse functional groups compared to traditional copolymerization approaches.
Key Steps: (1) P[E] was synthesized by ring-opening polymerization (ROP) of L-glutamic acid γ-benzyl ester N-carboxyanhydride (NCA) using n-hexylamine as an initiator (monomer/initiator ratio = 100) in 4 wt% 1,4-dioxane at 30°C for 72 hours, followed by benzyl deprotection with trifluoroacetic acid (TFA) and trifluoromethanesulfonic acid (TFMSA) (200 mg polymer in 4 mL TFA, 200 µL TFMSA, 4 hours at 22°C). (2) Carboxyl groups of P[E] were activated with N,N’-diisopropylcarbodiimide (DIC, 1.1-fold excess) and N-hydroxysuccinimide (NHS, 2-fold excess) in DMF (15 wt% polymer concentration) for 1 hour at 22°C. (3) A mixture of amino acids (basic:histidine:hydrophobic = 6:3.5:1.5, 110 mol% total) and D-glucosamine (60 mol%) was added, reacted for 4 hours in DMF/water (70/30 or 80/20 v/v), and purified by dialysis (MWCO 2000) against DMF, water, and 1 M NaCl. (4) Protective groups (e.g., Mtr, Trt, Boc) were removed using TFA/TFMSA. This stepwise approach ensured controlled modification while maintaining polymer integrity.
Data Collection and Analysis: Polymer composition was determined by hydrolyzing 2 mg of copolymer in 4 mL 6 M HCl (110°C, 4 days), followed by quantitative HPLC analysis of free amino acids and glucosamine using dansyl chloride derivatization (Shimadzu LC salicylate-30 Nexera, Kinetex C18 column, 254 nm detection). Molecular weight and dispersity of the intermediate P[E(OBzl)] were assessed via size-exclusion chromatography (SEC) in DMF with 0.1 M LiBr (Shimadzu LC-20, Styragel HMW6E column).
Table 1. Composition of the amphiphilic copolymers, as determined by quantitative HPLC amino acid analysis.
Figure 1. SEC trace of poly(α,L-glutamic acid γ-benzyl ester). Conditions: Styragel Column, HMW6E, Waters (7.8 mm × 300 mm, 15–20 µm bead size), DMF containing 0.1M LiBr, 40 °C, elution rate 0.3 mL/min, refractometric detection.
Results: HPLC revealed 69-80 mol% modification of carboxyl groups, with specific compositions (e.g., P[EE®E(H)E(F)E(Glc)]: 28% Arg, 16% His, 5% Phe, 20% Glc). SEC showed P[E(OBzl)] had an Mn of 14,400 Da and dispersity of 1.40, confirming a consistent starting material. These data validated successful functionalization.
Novel Aspects: Unlike traditional ROP-based synthesis, this post-modification approach allowed rapid, one-pot attachment of multiple functional groups, reducing labor and enabling tailored hydrophobicity and charge. This flexibility enhances adaptability over fixed-composition copolymerization methods for drug delivery applications.
Experiment 2: Preparation and Characterization of Nanoparticles
Primary Technique: Nanoparticle formation relied on self-assembly via slow gradient phase inversion (dialysis) for Phe- and Ile-containing polypeptides, and nanoprecipitation for Trp-containing polypeptides, chosen to optimize particle size and stability based on polymer solubility and hydrophobicity.
Key Steps: (1) For self-assembly, 2 mg/mL polymer in DMF was dialyzed (MWCO 2000) against water over 48 hours, freeze-dried, and redispersed in 0.01 M phosphate buffer (pH 7.4) at 2 mg/mL. (2) For nanoprecipitation, 2 mg polymer in 100 µL DMSO was added dropwise to 900 µL water/PBS under stirring (1000 rpm), ultrasonicated (30%, 30 s), and stabilized at 4°C for 30 minutes. (3) Particle size and charge were measured at 0.2 mg/mL in water. These methods were selected to ensure uniform nanoparticle formation suited to each polymer’s properties.
Data Collection and Analysis: Hydrodynamic diameter (DH) and polydispersity index (PDI) were measured using dynamic light scattering (DLS, ZetasizerNano-ZS, 633 nm, 173°) and nanoparticle tracking analysis (NTA, NanoSight NS300). Zeta potential was assessed via electrophoretic light scattering (ELS). Transmission electron microscopy (TEM, Jeol JEM-2100) with uranyl acetate staining quantified dry-state diameter from 30+ particles using ImageJ.
Table 2. Characteristics of nanoparticles obtained from the synthesized amphiphilic polypeptides.
Figure 2. TEM images of empty (a,c) and PTX‐loaded (b,d) nanoparticles based on P[EE®E(H)E(I)E(Glc)] (a,b) and P[EE(O)E(H)E(I)E(Glc)] (c,d
Results: Nanoparticles ranged from 200-350 nm (NTA) and 230-400 nm (DLS), with PDI of 0.10-0.35 and zeta potentials of -39.8 to -46.3 mV. TEM confirmed spherical morphology with dry diameters (e.g., 300 ± 52 nm for P[EE®E(H)E(I)E(Glc)]), 10-20% smaller than DLS values, indicating dense packing.
Novel Aspects: The dual-method approach (self-assembly vs. nanoprecipitation) tailored to polymer hydrophobicity produced stable, negatively charged nanoparticles with reduced macrophage uptake compared to positively charged traditional systems, enhancing circulation time for drug delivery.
Experiment 3: Paclitaxel Loading and Nanoformulation Characterization
Primary Technique: Paclitaxel (PTX) was encapsulated via a lyophilization-ultrasonication method, selected for its high encapsulation efficiency and ability to handle hydrophobic drugs, improving bioavailability over conventional solvent-based formulations.
Key Steps: (1) 50-200 µg PTX in 50-200 µL DMSO was mixed with 1 mg polymer in 400 µL DMSO, ultrasonicated (20 s), and lyophilized. (2) The lyophilizate was redispersed in 0.01 M PBS (pH 7.4) under ultrasonication (60 s) to 1 mg/mL. (3) Free PTX was quantified after ultrafiltration (MWCO 5000) by reversed-phase HPLC (Shimadzu LC-20AD, Grace Smart C18 column, 237 nm). This method ensured maximal drug incorporation and nanoparticle stability.
Data Collection and Analysis: Loading efficacy was calculated from HPLC data (gradient: 0-100% acetonitrile, 0-15 min). DH, PDI, and zeta potential of PTX-loaded nanoparticles were measured by DLS and ELS. TEM assessed morphology. Stability was monitored via DLS over 3 weeks at 20°C.
Results: Loading efficacy was 98-100% at 50 µg PTX/mg (5 wt%) and 80-92% at 200 µg/mg (16-18.4 wt%). PTX-loaded nanoparticles had DH of 170-290 nm (DLS), a 30-110 nm reduction from empty particles, with stable sizes over 3 weeks (except P[EE(O)E(F)E(H6-pept)]). TEM showed spherical particles (e.g., 210 ± 56 nm for P[EE®E(H)E(I)E(Glc)]).
Novel Aspects: The lyophilization method achieved higher PTX loading (up to 18.4 wt%) than many traditional nano-delivery systems (1.5-15 wt%), with size reduction due to hydrophobic interactions, offering superior drug capacity and stability for cancer therapy.
Experiment 4: Biological Evaluation of Nanoparticles and PTX Nanoformulations
Primary Technique: In vitro biological evaluation used cell viability (CTB assay), macrophage uptake (flow cytometry), and cytostatic activity (CTB assay on A549 cells), chosen to assess safety, stealth properties, and efficacy against lung cancer, aligning with PTX’s therapeutic targets.
Key Steps: (1) BEAS-2B cells (4 × 10³/well) were incubated with 4-1000 µg/mL nanoparticles for 72 hours, viability measured by CTB. (2) J774A.1 macrophages (4 × 10⁴/well) were incubated with Cy5-labeled nanoparticles (25 µg/mL, 5 µg Cy5/mg) for 5 hours, uptake quantified by flow cytometry (BD Accuri C6, 585/40 filter). (3) A549 cells (4 × 10³/well) were treated with 0.625-1000 ng/mL PTX (free or loaded) for 72 hours, IC50 calculated from CTB dose-response curves. These steps ensured comprehensive safety and efficacy profiling.
Data Collection and Analysis: Fluorescence (CTB, 560/590 nm) quantified viability. Cy5 fluorescence (650 nm) measured uptake, normalized to PLA nanoparticles. IC50 was derived via non-linear fitting (OriginPro 8.6). One-way ANOVA (p < 0.05) assessed significance.
Figure 3. Viability of BEAS‐2B cells in the presence of nanoparticles based on Phe‐ (a), Ile‐ (b) and Trp‐containing glycopolypeptides © as well as nanoparticles self‐assembled from a polypeptide modified with H6‐peptide (d) (CTB assay, 72 h)
Table 2. Cytostatic effect of the developed PTX nanoformulations (A549 cell line, 72 h).
Results: Nanoparticles showed >75% BEAS-2B viability up to 500 µg/mL, 28-45% macrophage uptake vs. PLA (100%), and PTX nanoformulations had IC50 of 1.3-7.8 ng/mL vs. 0.8 ng/mL (free PTX), indicating high efficacy with gradual release.
Novel Aspects: The negatively charged glycopolypeptide nanoparticles reduced macrophage uptake compared to cationic systems, enhancing stealth properties, while maintaining potent cytostatic effects, offering a safer, more effective alternative to traditional PTX formulations like PTX-LANS.
Conclusion
The successful development of this drug delivery system was achieved through the synthesis of amphiphilic (glyco)polypeptides via post-polymerization modification of poly(α,L-glutamic acid). This innovative approach allowed for the effective incorporation of various functional groups, resulting in nanoparticles that exhibit favorable characteristics for drug delivery applications.
The highlights of the study include the formation of nanoparticles with hydrodynamic diameters ranging from 170 to 330 nm, coupled with high paclitaxel loading efficiencies of 80-99%. Additionally, these nanoparticles demonstrated significant cytostatic efficacy against human lung adenocarcinoma cells (A549), with IC50 values ranging from 1.3 to 7.8 ng/mL, indicating strong anti-cancer potential. Importantly, the nanoparticles were non-toxic to normal lung epithelial cells (BEAS-2B) and exhibited reduced macrophage uptake, which enhances their potential as safe and effective delivery systems for hydrophobic drugs like paclitaxel. Overall, this research contributes to the advancement of drug delivery technologies, addressing the challenges associated with conventional methods and improving the therapeutic outcomes for lung cancer treatment.
Reference
Dzhuzha, Apollinariia Yu., et al. “Amphiphilic Polypeptides Obtained by the Post-Polymerization Modification of Poly(Glutamic Acid) and Their Evaluation as Delivery Systems for Hydrophobic Drugs.” International Journal of Molecular Sciences, vol. 24, no. 2, 2023, article 1049. MDPI, https://doi.org/10.3390/ijms24021049.