Lanthanide-based β-Tricalcium Phosphate Upconversion Nanoparticles as an Effective Theranostic Nonviral Vectors for Image-Guided Gene Therapy

Abstract

Lanthanide-based beta-tricalcium phosphate (β-TCP) upconversion nanoparticles are exploited as a non-viral vector for imaging guided-gene therapy by virtue of their unique optical properties and multi-modality imaging ability, high transfection efficiency, high biocompatibility, dispersibility, simplicity of synthesis and surface modification. Ytterbium and thulium-doped β-TCP nanoparticles (βTCPYbTm) are synthesized via co-precipitation method, coated with polyethylenimine (PEI) and functionalized with a nuclear-targeting peptide (TAT). Further, in vitro studies revealed that the nanotheranostic carriers are able to transfect cells with the plasmid eGFP at a high efficiency, with approximately 60% of total cells producing the fluorescent green protein. The optimized protocol developed comprises the most efficient βTCPYbTm/PEI configuration, the amount and the order of assembly of βTCPYbTm:PEI, TAT, plasmid DNA and the culturing conditions. With having excellent dispersibility and high chemical affinity toward nucleic acid, calcium ions released from βTCPYbTm:PEI nanoparticles can participate in delivering nucleic acids and other therapeutic molecules, overcoming the nuclear barriers and improving the transfection efficacy. Equally important, the feasibility of the upconversion multifunctional nanovector to serve as an effective contrast agent for imaging modality, capable of converting low-energy light to higher-energy photons via a multi-photons mechanism, endowing greater unique luminescent properties, was successfully demonstrated.

Keywords: NIR upconversion theranostic nanoparticles; PEI; TAT; gene therapy.; lanthanide-based β-TCP.

Figure 1

A) X-ray diffraction pattern of Yb/Tm-doped calcium phosphate nanoparticles as synthesized (CDHA phase) and treated at 900 °C for 3 min (β-TCP phase). B) TEM micrograph of bare βTCPYbTm nanoparticles.

Figure 2

A) Emission and excitation spectra of βTCPYbTm nanoparticles. Emission bands of Tm³⁺ excited by upconversion process through energy transfer from Yb³⁺, using pulsed laser at 973 nm. B) Schematic diagram of energy level transition of Yb³⁺ - Tm³⁺ ions in the upconversion process.

Figure 3

Upconversion nanoparticles internalization by L929 cell line, after 24h of incubation. The blue (A, λ_em ≈ 450-480 nm) and red (B, λ_em = 630-670 nm) nanoparticle emissions when excited at 975 nm, the nucleus in green (C, λ_ex = 488 nm), bright field (E) and merged channels (D and F). Scale bar = 75 μm.

Figure 4

L929 cells cultivated for 48 hours with 50 µL of nanoparticle solutions: (A) 50:1 w/w βTCPYbTm:PEI and B) 50 bare βTCPYbTm. Nanoparticles emission is represented in magenta dots and nuclei emission in green. Scale bar = 75 µm.

Figure 5

Gel retardation - eGFP-C1 plasmid DNA binding assay. Lane 1: ladder 1000bp; Lane 2: pDNA only; Lane 3: 12.5:1 w/w βTCPYbTm:PEI pH 7.3 + pDNA; Lane 4: 25:1 w/w βTCPYbTm:PEI pH 7.3 + pDNA; Lane 5: 50:1 w/w βTCPYbTm:PEI pH 7.3 + pDNA; Lane 6: 250:1 w/w βTCPYbTm:PEI pH 7.3 + pDNA; Lane 7: 250 bare βTCPYbTm + pDNA; Lane 8: 12.5:1 w/w βTCPYbTm:PEI pH 5.2 + pDNA; Lane 9: 50:1 w/w βTCPYbTm:PEI pH 5.2 + pDNA; Lane 10: 50 bare βTCPYbTm + pDNA; Lane 11: PEI only + pDNA; Lane 12: ladder 1000bp. The pDNA remains unbound only for bare βTCPYbTm suspensions (lanes 7 and 10).

Figure 6

HEK293T cells transfected with [(50 µL TAT + 600 ng pDNA) + 10 µL βTCPYbTm:PEI] in DMEM medium containing: A) free-antibiotic (0% penicillin and streptomycin) and -serum (0% FBS); B) 1% penicillin and streptomycin and 0% FBS; C) 1% penicillin and streptomycin and 1% FBS; and D) 1% penicillin and streptomycin and 10% FBS. Scale bar = 300 μm.

Figure 7

TEM analysis of [βTCPYbTm:PEI + (DNA + TAT)]. Scale bar = 200 nm

Figure 8

Transfection efficiency of PEI only versus βTCPYbTm:PEI nanocarriers, showing that lanthanide-based calcium phosphate nanoparticles, in the presence or absence of TAT peptide, improved the percentage of transfected cells; *p < 0.05 and ** p < 0.01.

Figure 9

Cell viability test. For the cytotoxicity evaluation, using the MTS assay, the HEK293T cells were incubated for 24 hours with the nanocarriers in the presence and absence of the pDNA (* p < 0.05). (βYTPEI abbreviation stands for βTCPYbTm:PEI).

Figure 10

Transfected cells with A/D) 35 µL of PEI only + 3.5 µg pDNA; B/E) 35 µL of βTCPYbTm:PEI + 3.5 µg pDNA and C/F) [20 µL βTCPYbTm + (150 µL TAT + 1.5 µg pDNA)], imaged 48 h post-transfected. Green emission represents GFP (λ_ex = 488 nm); magenta dots are the upconversion nanoparticles (λ_ex = 975 nm) and blue emission are the nucleus stained with DAPI (λ_ex = 405 nm). Scale bar = 75 µm.

Figure 11

HEK293T cells transfected with [20µL βTCPYbTm + (150 µL TAT + 1.5 µg pDNA)], imaged 1 h (A), 6 h (B) and 24 h (C) post-transfection. Green emission represents GFP (λ_ex = 488 nm); magenta dots are the upconversion nanoparticles (λ_ex = 975 nm) and blue emission are the nucleus stained with DAPI (λ_ex = 405nm). Scale bar = 75 µm.

Figure 12

HEK293T cells transfected with [20µL βTCPYbTm:PEI + (150 µL TAT-TAMRA + 1.5 µg eGFP] for overnight analysis from 6 to 24 h post-transfection. Images were acquired every 30 min by the LSM 880 multiphoton inverted confocal microscope. Samples were excited at λ_ex = 975 nm (blue emission - nanoparticles), λ_ex = 488 nm (green emission - GFP) and λ_ex = 561 nm (red emission -TAT-TAMRA). Scale bar = 75 µm.