Multi-functionalized carbon dots as theranostic nanoagent for gene delivery in lung cancer therapy

Abstract

Theranostics, an integrated therapeutic and diagnostic system, can simultaneously monitor the real-time response of therapy. Different imaging modalities can combine with a variety of therapeutic moieties in theranostic nanoagents. In this study, a multi-functionalized, integrated theranostic nanoagent based on folate-conjugated reducible polyethylenimine passivated carbon dots (fc-rPEI-Cdots) is developed and characterized. These nanoagents emit visible blue photoluminescence under 360 nm excitation and can encapsulate multiple siRNAs (EGFR and cyclin B1) followed by releasing them in intracellular reductive environment. In vitro cell culture study demonstrates that fc-rPEI-Cdots is a highly biocompatible material and a good siRNA gene delivery carrier for targeted lung cancer treatment. Moreover, fc-rPEI-Cdots/pooled siRNAs can be selectively accumulated in lung cancer cells through receptor mediated endocytosis, resulting in better gene silencing and anti-cancer effect. Combining bioimaging of carbon dots, stimulus responsive property, gene silencing strategy, and active targeting motif, this multi-functionalized, integrated theranostic nanoagent may provide a useful tool and platform to benefit clinicians adjusting therapeutic strategy and administered drug dosage in real time response by monitoring the effect and tracking the development of carcinomatous tissues in diagnostic and therapeutic aspects.

Figure 1. Synthesis route of fc-rPEI-Cdots/siRNA multifunctional nanoagents.

In this study, fc-rPEI-Cdots nanoagent were synthesized through a serial process. Microwave pyrolysis was first carried out to fabricate PEI-Cdots. Then, rPEI-Cdots were formed by conjugation of excess PEI molecules to PEI-Cdots through Michael addition. Afterwards, folic acids were attached to rPEI-Cdots to develop fc-rPEI-Cdots nanoagnet. With the possitive charge on the surface, our fc-rPEI-Cdots naonagent can compex with negatively charged therapeutic siRNA molecules.

Figure 2

Characterization of fc-rPEI-Cdots by (A) H¹NMR and (B) FT-IR. From H¹NMR, disappearance of alkenyl proton peaks at 5.6 and 6.2 ppm (peak a and b) and preservation of peak at 2.8 ppm (peak d) proved successful synthesis of rPEI-Cdots. Appearance of proton resonance peak between 1.8 ppm showed conjugations of folate. From FT-IR, preservation of C-N and N-H stretching signals of PEI-Cdots (ν = 1018 cm⁻¹and 3378 cm⁻¹), amide C = O stretching (ν = 1684 cm⁻¹) signal of BAC, and appearance of phenyl absorption peak of folic acid at 1470 cm⁻¹ and 1513 cm⁻¹ in the spectrum of fc-rPEI-Cdots demonstrated successful synthesis of fc-rPEI-Cdots.

Figure 3

TEM images of (A) rPEI-Cdots and (B) fc-rPEI-Cdots. rPEI-Cdots and fc-rPEI-Cdots were well dispersed in aqueous solution. The particle size of rPEI-Cdots and fc-rPEI-Cdots was below 200nm. (C) The higher magnification image of fc-rPEI-Cdots in selected area.

Figure 4. Diagnostic property of fc-rPEI-Cdots nanoparticles.

(A) Absorption and photoluminescence spectrum of rPEI-Cdots, PEI-Cdots, folate, and fc-rPEI-Cdots. Insert: Photoluminescence (PL) of rPEI-Cdots and fc- rPEI-Cdots under UV excitation. Strongest photoluminescence of fc-rPEI-Cdots appeared at 460 nm when excited by 360 nm. (B) The PL intensity of rPEI-Cdots and fc-rPEI-Cdots in different pH environment (pH 5, 8). (C) Enhanced accumulation of fc-rPEI-Cdots in H460 could be observed by fluorescent microscopy. (D) fc-rPEI-Cdots (blue) and FAM labeled-siRNA (green) were delivered intracellularly.

Figure 5

Electrophoresis of fc-rPEI-Cdots/siRNA nanocomplex with different weight ratio of 5, 10, 15, and 20 under normal (without DTT treatment (A)) and reductive environment (with 25mM DTT reducing agent treated for 4 hours at 37 °C (B)). The encapsulated siRNA could complex with fc-rPEI-Cdots compactly at weight ratio above 15 and be released in reductive environment. (Weight ratio is defined as the weight of nanoparticles versus the weight of siRNA. The weight of siRNA can be derived and calculated from the measurement of OD value).

Figure 6. Biocompatibility and therapeutic effect of fc-rPEI-Cdots/siRNA complexes.

(A) Biocompatibility of fc-rPEI-Cdots in human lung cancer cells and mouse embryo fibroblasts after incubated with fc-rPEI-Cdots for 24 hours. Viability of each group remained above 90% suggests that fc-rPEI-Cdots is a non-cytotoxic and biocompatible material. (B) Viability of lung cancer cells (H460) after treated with fc-rPEI-Cdots/single siRNA (cyclin B1) and rPEI-Cdots/pooled siRNA (cyclin B1 + EGFR) for 24 hours, 48 hours, and 72 hours. Viability of H460 dropped to 30% after treated with fc-rPEI-Cdots/pooled siRNA after 72 hours. (*p < 0.05 ; **p < 0.01, N = 4).

Figure 7. Gene silencing effect of EGFR and Cyclin B1 after treated with fc-rPEI-Cdots/pooled siRNA, fc-rPEI-Cdots/single siRNA, and pooled siRNA (without nanocarrier) in H460 for 12 hours, 24 hours, and 48 hours.

The group of pooled siRNA showed transient gene silencing effect in 12 hours, and failed to reduce gene expression thereafter. On the other hand, fc-rPEI-Cdots/pooled siRNA showed sustained gene silencing effect for prolong period of time in 48 hrs. (N = 4).

Figure 8. Monitoring luciferase inhibition in vivo with bioluminescent imaging.

Representative images show the reduction in lung tumor size following intratracheal instillation of fc-rPEI-Cdots nanoagents in luciferase-expressing H460 lung carcinoma. Panels (A–C) depict bioluminescent images of the lungs before and after treatment. (B) 7 days, (C) 10 days after two times inhaled administration. After aerosol delivery, the fc-rPEI-Cdots/pooled siRNA nanoagents (D) can accumulated at lung region when compared to PBS (E).