Near-infrared Light Triggered Photodynamic Therapy In Combination With Gene Therapy Using Upconversion Nanoparticles For Effective Cancer Cell Killing

Objective To study the dual-polymer coated upconversion nanoparticles(UCNPPEG@2×PEI) synthesized via a layer by layer strategy for near-infrared light triggered photodynamic therapy.Methods 1) Synthesize Gd3+-doped Na Gd F4(Gd : Yb : Er =78% : 20% : 2%) UCNPs nanoparticles using a high temperature thermal decomposition method, which was then functionalized by polyethylene glycol,polyethylenimine and poly acrylic acid layer by layer to obtain excellent water solubility solution. Next, the morphology of UCNPs and zeta potentials of different layers of UCNP-PEG@2×PEI were assessed. 2) Chlorin e6(Ce6) molecules were loaded onto UCNPs via hydrophobic interactions. The loading capacity of Ce6 and the release of Ce6 from the UCNP-PEG@2×PEI- Ce6 nanocomplex were then evaluated. And the UV-Vis-NIR absorbance spectra of UCNP-PEG@2×PEI-Ce6 were measured. The singlet oxygen produced by UCNP- PEG@2×PEI-Ce6 complex was determined. 3) The MR T1-weighted images were performed using a series of UCNP-PEG@2×PEI-Ce6 solutions with different concentrations. 4) The cytotoxicity of UCNP-PEG@ 2×PEI-Ce6 was evaluated by MTT assay. 5) UCNP-PEG@2×PEI-Ce6 solutions with different concentrations incubated with He La cells for 4 h. Upon 980 nm laser irradiation 20 minutes, cell viability assays were conducted for He La cells.Results 1) The transmission electron microscopy(TEM) imaging showed that the synthesized UCNPs nanoparticles were monodispersed nanocrystals with a uniform average diameter of ~ 30 nm. UCNP-PEG@2×PEI exhibited well stability in physiological solutions after different layers of modification and excellent positive charges after the second layer of PEI was coated. 2) The amount of Ce6 loaded onto UCNPs reached 10–11% when the UCNP–Ce6(w/w) ratio was 2:1. Further increase of Ce6 loading, however, would result in decreased stability of those nanoparticles. 3) The UCL intensity of UCNP-PEG@2×PEI was still high. However, an obvious decrease of upconversion luminescence(UCL) intensity of UCNPs after Ce6 loading was observed, particularly for its emission peak at 660 nm, which overlapped with the absorbance peak of Ce6 at the same wavelength. 4) Effective generation of singlet oxygen(SO) by UCNP-PEG@ 2×PEI-Ce6 under 980 nm laser irradiation could be detected. In contrast, free Ce6 under excitation by such NIR light was not able to produce SO. 5) MR images showed UCNP-PEG@2×PEI-Ce6 could serve as imaging probes for MR imaging and showed a concentration-dependent brightening effect. 6) In vitro cytotoxicity assay showed no appreciate cytotoxic response of cells even UCNP-PEG@2×PEI-Ce6 at high concentrations. 7) Upon 980 nm laser irradiation, the cell viabilities gradually decreased with the rise of UCNP-PEG@2×PEI-Ce6 concentrations, indicating the effectiveness of NIR-induced PDT mediated by UCNPs.Conclusion UCNP-PEG@2×PEI which with two layers PEI coated on UCNPs were stable in physiological environments, exhibited excellent positive charges and high UCL intensity. The binding of Ce6 onto UCNPs happens mainly via hydrophobic interactions between Ce6 molecules and the hydrophobic oleic acid layer on the surface of UCNP cores beneath the PEG/PEI coating. In vitro study,the as-made UCNP-PEG@2×PEI-Ce6 nanoparticles uncovered no apparent toxicity. Besides, UCNP-PEG@2×PEI-Ce6 showed excellent capacity of MR imaging. In this system, UCNPs with the unique upconversion photoluminescence could serve as an imaging probe, as well as an energy donor useful in triggering photodynamic therapy(PDT) under near-infrared(NIR) light, which offers greatly improved tissue penetration. Therefore, UCNP-PEG@2×PEI-Ce6 could serve as novel imaging-trackable nano-vectors for tumor diagnosis and treatment.Objective Study of the multi-layer dual-polymer coated upconversion nanoparticles serve as gene delivery vectors and NIR-triggered PDT in combination with gene therapy for effective cancer cell killing.Methods 1) To study the binding ability of UCNP-PEG@2×PEI-Ce6 and siRNA, gel electrophoresis assay was conducted. Appropriate amounts of UCNP-PEG@2×PEI- Ce6 were mixed with 1 mg si RNA at different N/P ratios(5, 10, 15, 20), after incubation for 20 min at room temperature, the samples were analyzed by 0.8% agarose gel electrophoresis. 2) In vitro si Plk1 transfection experiment, appropriate amounts of UCNP-PEG@ 2×PEI-Ce6 were mixed with si Plk1 at different N/P ratios and incubated for 20 min at room temperature before being added into He La cells with or without the serum. 3) We used a confocal fluorescence microscope to study the cellular uptake of UCNP-PEG@2×PEI-Ce6/si RNA complex and checked the serum effects on the si RNA transfection by using fluorescently labeled si RNA(FAM-si RNA). 4) Western blotting and RT-q PCR experiments were carried out to determine the expression levels of Plk1 and the Plk1 m RNA after UCNP-induced si RNA transfection at different N/P ratios with or without FBS. 5) UCNP-PEG@2× PEI-Ce6/si RNA treated He La under the optimized transfection conditions were treated with or without 980 nm laser irradiation. The Plk1 protein levels in those samples were measured by Western blotting to check whether the production of singlet oxygen during PDT would affect si RNA loaded on UCNPs. 6) Cell viability assays were performed for He La cells after various treatments including PDT, gene therapy and combined PDT/gene therapy. Microscopy images of calcein-AM & PI double stained(living & dead cells) He La cells confirmed the result.Results 1) At N/P ratios over 10, the majority of si RNA were effectively loaded onto nanoparticles. 2) Confocal fluorescence microscope images showed that both Ce6 and si RNA were successfully shuttled into He La cells by UCNPs and the si RNA delivery ability of UCNP-PEG@2×PEI-Ce6 was increased by the serum in the transfection medium. 3) Western blotting and RT-q PCR results indicate that the serum-containning medium could enhance the siRNA-induced down-regulation of Plk1 protein expression. 4) The Plk1 protein levels measured by western blotting demonstrates that PDT, which although could produce cytotoxic singlet oxygen, would not affect the si RNA-induced gene silencing, making the combination of PDT with gene therapy a reasonable approach. 5) The combined photodynamic and gene therapy offered a significantly higher cancer cell killing effect compared to mono-therapy by either gene therapy or PDT alone.Conclusion UCNPs with the unique upconversion photoluminescence could serve as an energy donor useful in triggering PDT under NIR light, which offers greatly improved tissue penetration. With well-engineered surface coatings, these nanoparticles appear to be an effective si RNA delivery vector that works well in the presence of serum. A new multifunctional UCNP-based nano-platform with well-designed surface is successfully developed for combined PDT and gene therapy of cancer.