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pH-responsive Virus-like Nanoparticles with Enhanced Tumour-targeting Ligands for Cancer Drug Delivery

View Article: PubMed Central - PubMed

ABSTRACT

Multifunctional nanocarriers harbouring specific targeting moieties and with pH-responsive properties offer great potential for targeted cancer therapy. Several synthetic drug carriers have been studied extensively as drug delivery systems but not much information is available on the application of virus-like nanoparticles (VLNPs) as multifunctional nanocarriers. Here, we describe the development of pH-responsive VLNPs, based on truncated hepatitis B virus core antigen (tHBcAg), displaying folic acid (FA) for controlled drug delivery. FA was conjugated to a pentadecapeptide containing nanoglue bound on tHBcAg nanoparticles to increase the specificity and efficacy of the drug delivery system. The tHBcAg nanoparticles loaded with doxorubicin (DOX) and polyacrylic acid (PAA) demonstrated a sustained drug release profile in vitro under tumour tissue conditions in a controlled manner and improved the uptake of DOX in colorectal cancer cells, leading to enhanced antitumour effects. This study demonstrated that DOX-PAA can be packaged into VLNPs without any modification of the DOX molecules, preserving the pharmacological activity of the loaded DOX. The nanoglue can easily be used to display a tumour-targeting molecule on the exterior surface of VLNPs and can bypass the laborious and time-consuming genetic engineering approaches.

No MeSH data available.


Conjugation of folic acid to tHBcAg nanoparticles using the nanoglue concept.(a) An SDS-polyacrylamide gel of the tHBcAg nanoparticles cross-linked with the pentadecapeptide containing the nanoglue. Lane M, molecular mass markers (kDa); lane 1, tHBcAg; lane 2, tHBcAg plus cross-linkers; and lane 3, tHBcAg plus the pentadecapeptide and cross-linkers. The arrow shows a shifted band of approximately 1 kDa above the 17 kDa tHBcAg. (b) Conjugation of folic acid (FA) to tHBcAg. Spectra of FA, tHBcAg nanoparticles (tHBcAg), tHBcAg nanoparticles cross-linked with pentadecapeptide (N-tHBcAg), FA-conjugated tHBcAg nanoparticles (FA-tHBcAg), and FA-conjugated tHBcAg nanoparticles using the nanoglue (FA-N-tHBcAg). (c) Electron micrographs of tHBcAg nanoparticles. Nanoparticles formed by tHBcAg (i), N-tHBcAg (ii), FA-tHBcAg (iii), and FA-N-tHBcAg (iv) were stained with uranyl acetate and observed via TEM. White bars indicate 50 nm.
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f2: Conjugation of folic acid to tHBcAg nanoparticles using the nanoglue concept.(a) An SDS-polyacrylamide gel of the tHBcAg nanoparticles cross-linked with the pentadecapeptide containing the nanoglue. Lane M, molecular mass markers (kDa); lane 1, tHBcAg; lane 2, tHBcAg plus cross-linkers; and lane 3, tHBcAg plus the pentadecapeptide and cross-linkers. The arrow shows a shifted band of approximately 1 kDa above the 17 kDa tHBcAg. (b) Conjugation of folic acid (FA) to tHBcAg. Spectra of FA, tHBcAg nanoparticles (tHBcAg), tHBcAg nanoparticles cross-linked with pentadecapeptide (N-tHBcAg), FA-conjugated tHBcAg nanoparticles (FA-tHBcAg), and FA-conjugated tHBcAg nanoparticles using the nanoglue (FA-N-tHBcAg). (c) Electron micrographs of tHBcAg nanoparticles. Nanoparticles formed by tHBcAg (i), N-tHBcAg (ii), FA-tHBcAg (iii), and FA-N-tHBcAg (iv) were stained with uranyl acetate and observed via TEM. White bars indicate 50 nm.

Mentions: To increase the accessibility of FA to the FR, nanoglue was used to display FA molecules at the spikes of tHBcAg nanoparticles (Fig. 1). A pentadecapeptide (KKKGGGSLLGRMKGA) containing 3 Lys residues at the N-terminus was synthesised and cross-linked to tHBcAg nanoparticles using Sulfo-NHS and EDC. The tHBcAg monomer shifted approximately 1 kDa on an SDS-polyacrylamide gel, demonstrating that the peptide was cross-linked to the monomer (Fig. 2a). The cross-linked tHBcAg nanoparticles were then conjugated with FA by using Sulfo-NHS and EDC, and the absorbance at wavelength 240–700 nm was measured. Conjugation of FA to tHBcAg using the nanoglue concept (FA-N-tHBcAg) exhibited a greater A360 value compared with conjugation of FA to tHBcAg (FA-tHBcAg) directly (Fig. 2b). The conjugation efficiency (CE) of FA was 6.1 ± 0.3% and 12.4 ± 0.5%, amounting to approximately 461 and 953 FA molecules conjugated to each FA-tHBcAg and FA-N-tHBcAg nanoparticle, respectively. FA:tHBcAg stoichiometry was determined to be 2:1 for FA-tHBcAg and 4:1 for FA-N-tHBcAg. This is because FA can be conjugated to the Lys residues of the pentadecapeptide and tHBcAg nanoparticles. Transmission electron micrographs showed that cross-linking of the nanoglue followed by conjugation of FA did not have an adverse effect on the spherical structure of the tHBcAg nanoparticles (Fig. 2c). To verify that FA molecules were indeed conjugated to Lys residues at the N-terminus of the pentadecapeptide, Lys-7 and Lys-96 of tHBcAg were first conjugated with NHS-fluorescein. The fluorescein-labelled tHBcAg nanoparticles (ftHBcAg) were then cross-linked with the pentadecapeptide and conjugated with FA (Supplementary Figure S1). A360 showed that FA was conjugated to the pentadecapeptide, and A500 demonstrated that tHBcAg was cross-linked with fluorescein molecules.


pH-responsive Virus-like Nanoparticles with Enhanced Tumour-targeting Ligands for Cancer Drug Delivery
Conjugation of folic acid to tHBcAg nanoparticles using the nanoglue concept.(a) An SDS-polyacrylamide gel of the tHBcAg nanoparticles cross-linked with the pentadecapeptide containing the nanoglue. Lane M, molecular mass markers (kDa); lane 1, tHBcAg; lane 2, tHBcAg plus cross-linkers; and lane 3, tHBcAg plus the pentadecapeptide and cross-linkers. The arrow shows a shifted band of approximately 1 kDa above the 17 kDa tHBcAg. (b) Conjugation of folic acid (FA) to tHBcAg. Spectra of FA, tHBcAg nanoparticles (tHBcAg), tHBcAg nanoparticles cross-linked with pentadecapeptide (N-tHBcAg), FA-conjugated tHBcAg nanoparticles (FA-tHBcAg), and FA-conjugated tHBcAg nanoparticles using the nanoglue (FA-N-tHBcAg). (c) Electron micrographs of tHBcAg nanoparticles. Nanoparticles formed by tHBcAg (i), N-tHBcAg (ii), FA-tHBcAg (iii), and FA-N-tHBcAg (iv) were stained with uranyl acetate and observed via TEM. White bars indicate 50 nm.
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f2: Conjugation of folic acid to tHBcAg nanoparticles using the nanoglue concept.(a) An SDS-polyacrylamide gel of the tHBcAg nanoparticles cross-linked with the pentadecapeptide containing the nanoglue. Lane M, molecular mass markers (kDa); lane 1, tHBcAg; lane 2, tHBcAg plus cross-linkers; and lane 3, tHBcAg plus the pentadecapeptide and cross-linkers. The arrow shows a shifted band of approximately 1 kDa above the 17 kDa tHBcAg. (b) Conjugation of folic acid (FA) to tHBcAg. Spectra of FA, tHBcAg nanoparticles (tHBcAg), tHBcAg nanoparticles cross-linked with pentadecapeptide (N-tHBcAg), FA-conjugated tHBcAg nanoparticles (FA-tHBcAg), and FA-conjugated tHBcAg nanoparticles using the nanoglue (FA-N-tHBcAg). (c) Electron micrographs of tHBcAg nanoparticles. Nanoparticles formed by tHBcAg (i), N-tHBcAg (ii), FA-tHBcAg (iii), and FA-N-tHBcAg (iv) were stained with uranyl acetate and observed via TEM. White bars indicate 50 nm.
Mentions: To increase the accessibility of FA to the FR, nanoglue was used to display FA molecules at the spikes of tHBcAg nanoparticles (Fig. 1). A pentadecapeptide (KKKGGGSLLGRMKGA) containing 3 Lys residues at the N-terminus was synthesised and cross-linked to tHBcAg nanoparticles using Sulfo-NHS and EDC. The tHBcAg monomer shifted approximately 1 kDa on an SDS-polyacrylamide gel, demonstrating that the peptide was cross-linked to the monomer (Fig. 2a). The cross-linked tHBcAg nanoparticles were then conjugated with FA by using Sulfo-NHS and EDC, and the absorbance at wavelength 240–700 nm was measured. Conjugation of FA to tHBcAg using the nanoglue concept (FA-N-tHBcAg) exhibited a greater A360 value compared with conjugation of FA to tHBcAg (FA-tHBcAg) directly (Fig. 2b). The conjugation efficiency (CE) of FA was 6.1 ± 0.3% and 12.4 ± 0.5%, amounting to approximately 461 and 953 FA molecules conjugated to each FA-tHBcAg and FA-N-tHBcAg nanoparticle, respectively. FA:tHBcAg stoichiometry was determined to be 2:1 for FA-tHBcAg and 4:1 for FA-N-tHBcAg. This is because FA can be conjugated to the Lys residues of the pentadecapeptide and tHBcAg nanoparticles. Transmission electron micrographs showed that cross-linking of the nanoglue followed by conjugation of FA did not have an adverse effect on the spherical structure of the tHBcAg nanoparticles (Fig. 2c). To verify that FA molecules were indeed conjugated to Lys residues at the N-terminus of the pentadecapeptide, Lys-7 and Lys-96 of tHBcAg were first conjugated with NHS-fluorescein. The fluorescein-labelled tHBcAg nanoparticles (ftHBcAg) were then cross-linked with the pentadecapeptide and conjugated with FA (Supplementary Figure S1). A360 showed that FA was conjugated to the pentadecapeptide, and A500 demonstrated that tHBcAg was cross-linked with fluorescein molecules.

View Article: PubMed Central - PubMed

ABSTRACT

Multifunctional nanocarriers harbouring specific targeting moieties and with pH-responsive properties offer great potential for targeted cancer therapy. Several synthetic drug carriers have been studied extensively as drug delivery systems but not much information is available on the application of virus-like nanoparticles (VLNPs) as multifunctional nanocarriers. Here, we describe the development of pH-responsive VLNPs, based on truncated hepatitis B virus core antigen (tHBcAg), displaying folic acid (FA) for controlled drug delivery. FA was conjugated to a pentadecapeptide containing nanoglue bound on tHBcAg nanoparticles to increase the specificity and efficacy of the drug delivery system. The tHBcAg nanoparticles loaded with doxorubicin (DOX) and polyacrylic acid (PAA) demonstrated a sustained drug release profile in vitro under tumour tissue conditions in a controlled manner and improved the uptake of DOX in colorectal cancer cells, leading to enhanced antitumour effects. This study demonstrated that DOX-PAA can be packaged into VLNPs without any modification of the DOX molecules, preserving the pharmacological activity of the loaded DOX. The nanoglue can easily be used to display a tumour-targeting molecule on the exterior surface of VLNPs and can bypass the laborious and time-consuming genetic engineering approaches.

No MeSH data available.