Glycopolymer-Cell-Penetrating Peptide (CPP) Conjugates for Efficient Epidermal Growth Factor Receptor (EGFR)
Yi-Yang Peng1, Haimei Hu2,3, Diana Diaz-Dussan1
1Department of Chemical & Materials Engineering, University of Alberta, Edmonton T6G 1H9, Alberta Canada.
Abstract:
Overexpression of epidermal growth factor receptor (EGFR) is observed in multiple cancers such as colorectal, lung, and cervical solid tumors. Regulating the EGFR expression is an efficient strategy to manage these malignancies, and it can be achieved by using short interfering RNA (siRNA). Cell-penetrating peptides (CPPs) demonstrated an excellent capability to enhance the cellular uptake of siRNA, but high knockdown efficiencies have not been achieved due to endosomal entrapment. In this work, Schiff's base reaction was used to modify a block {P[LAEMA(2-lactobionamidoethyl methacrylamide)37]-b-P[FPMA(4-formyl phenyl methacrylate)2-st-DMA(N,N-dimethylacrylamide)2], P2} and two statistical [P(LAEMA23-st-FPMA3) (P3) and P(LAEMA25-st-FPMA2-st-DMA2) (P4)] aldehyde-based and galactose-based polymers, prepared via reversible addition-fragmentation chain-transfer (RAFT) polymerization. An arginine-rich peptide (ARP, KRRKRRRRRK) was used as a cell-penetrating peptide (CPP) and conjugated to the polymers via a Schiff base reaction. The resulting glycopolymer-peptide conjugates were utilized to condense the siRNA to prepare polyplexes with multivalent CPPs (MCPPs, a nanoparticle with multiple copies of the CPP) to enhance the endosomal escape. The polyplexes have different surface properties as determined by the architecture of polymers and the insertion of dimethyl amide moieties. The enhancement of cellular internalization of ARP was observed by labeling the polyplexes with fluorescein isothiocyanate (FITC)-siRNA showing a localization of polyplexes in the cytoplasm of a HeLa (cervical cancer) cell line. In the in vitro EFGR silencing study, the statistical glycopolymer-peptide (P3-P) polyplexes had superior EGFR silencing efficiency in comparison with the other polymers that were studied. Furthermore, P3-P polyplexes led to less off-targeting silencing than lipofectamine 3000. These encouraging results confirmed the potency of decorating galactose-based polymers with CPP, like ARP for their application in siRNA delivery and management of cervical carcinomas.
Insights
This study developed novel galactose-based glycopolymer-peptide nanoparticles for enhanced siRNA delivery. These nanoparticles effectively silenced epidermal growth factor receptor (EGFR) in cervical cancer cells, showing promise for cancer therapy.
Area of Science:
- Biomaterials Science
- Nanotechnology
- Cancer Therapeutics
Background:
- Overexpression of epidermal growth factor receptor (EGFR) drives various cancers, including cervical carcinoma.
- Short interfering RNA (siRNA) offers a strategy to regulate EGFR expression for cancer management.
- Existing cell-penetrating peptides (CPPs) enhance siRNA uptake but struggle with endosomal entrapment, limiting knockdown efficiency.
Purpose of the Study:
- To develop novel glycopolymer-peptide nanoparticles for efficient siRNA delivery and endosomal escape.
- To investigate the potential of these nanoparticles for targeted EGFR silencing in cervical cancer.
- To evaluate the efficacy and specificity of the developed siRNA delivery system compared to commercial alternatives.
Main Methods:
- Synthesis of galactose-based aldehyde-containing polymers (P2, P3, P4) via RAFT polymerization.
- Conjugation of an arginine-rich peptide (ARP) to polymers using Schiff base reaction, forming glycopolymer-peptide conjugates.
- Preparation of multivalent CPP polyplexes by condensing siRNA with the conjugates.
- Evaluation of cellular internalization using FITC-labeled siRNA in HeLa cells.
- Assessment of EGFR silencing efficiency and off-targeting effects in vitro.
Main Results:
- The glycopolymer-peptide conjugates successfully formed polyplexes with siRNA, enhancing cellular internalization and endosomal escape.
- Statistical glycopolymer-peptide (P3-P) polyplexes demonstrated superior EGFR silencing efficiency in HeLa cells.
- P3-P polyplexes exhibited reduced off-targeting effects compared to Lipofectamine 3000.
- The study confirmed enhanced cytoplasmic localization of polyplexes.
Conclusions:
- Galactose-based glycopolymer-peptide nanoparticles are effective carriers for siRNA delivery.
- The developed system shows significant potential for targeted EGFR silencing in cervical cancer therapy.
- This approach offers a promising strategy for improving siRNA delivery efficiency and specificity in cancer treatment.
