A biomimetic hybrid nanoplatform for encapsulation and precisely controlled delivery of theranostic agents. [Corrected]

Abstract

Nanoparticles have demonstrated great potential for enhancing drug delivery. However, the low drug encapsulation efficiency at high drug-to-nanoparticle feeding ratios and minimal drug loading content in nanoparticle at any feeding ratios are major hurdles to their widespread applications. Here we report a robust eukaryotic cell-like hybrid nanoplatform (EukaCell) for encapsulation of theranostic agents (doxorubicin and indocyanine green). The EukaCell consists of a phospholipid membrane, a cytoskeleton-like mesoporous silica matrix and a nucleus-like fullerene core. At high drug-to-nanoparticle feeding ratios (for example, 1:0.5), the encapsulation efficiency and loading content can be improved by 58 and 21 times, respectively, compared with conventional silica nanoparticles. Moreover, release of the encapsulated drug can be precisely controlled via dosing near infrared laser irradiation. Ultimately, the ultra-high (up to ∼87%) loading content renders augmented anticancer capacity both in vitro and in vivo. Our EukaCell is valuable for drug delivery to fight against cancer and potentially other diseases.

Figure 1. Synthesis and characterization of biomimetic hybrid nanoparticles.

(a) A schematic illustration of the procedure for preparing fullerene- (C60) embedded silica (C60S) nanoparticles, modifying the C60S nanoparticles with APTMS to form C60S-A nanoparticles, and further coating the C60S-A nanoparticles with phospholipids (DPPC) to produce LC60S nanoparticles. (b) Transmission electron microscopy images of the C60S and LC60S nanoparticles. Also shown is a zoom-in and schematic view of the LC60S nanoparticles to illustrate their eukaryotic cell-like configuration with a phospholipid membrane, a cytoskeleton of mesoporous silica and a nucleus of fullerene. (c) Scanning electron microscopy images of the C60S and LC60S nanoparticles showing their homogeneous size distribution. (d) The difference in surface zeta potential of C60S, C60S-A and LC60S nanoparticles indicating the successful coating of first positively charged APTMS and then negatively charged phospholipid on the surface of C60S nanoparticles. (e) Size distribution of C60S nanoparticles showing they form aggregates in both cell culture medium and the blood. (f) Surface zeta potential of C60S and LC60S nanoparticles in medium (C60S, M, and LC60S, M) and the blood (C60S, B, and LC60S, B) showing the LC60S nanoparticles stay negatively charged, whereas the C60S nanoparticles become nearly neutral. (g) Size distribution of LC60S nanoparticles showing their stability in water, culture medium and the blood. APTMS, (3-aminopropyl) trimethoxysilane; TEOS, tetraethyl orthosilicate.

Figure 2. Encapsulation and controlled release of theranostic agents.

(a) Encapsulation efficiency (EE) of doxorubicin hydrochloride (DOX) and indocyanine green (ICG) by mixing the agents with nanoparticles for different times. The LC60S nanoparticles could encapsulate DOX efficiently but not ICG by simply mixing. However, by loading DOX first, the resultant DOX-laden LC60S (LC60S-D) nanoparticles could encapsulate ICG with an EE of ∼100% by simply mixing for only 30 min. (b) EE and (c) loading content (LC) of DOX in LC60S and silica (SiO₂) nanoparticles at different feeding ratios by simply mixing for 30 min and 24 h, showing the remarkably improved EE and LC of DOX in LC60S nanoparticles compared with the conventional SiO₂ nanoparticles. (d) The EE of ICG when mixed with LC60S-D nanoparticles at different ratios of DOX to ICG showing its dependence on the DOX content in the nanoparticles. (e) The total LC of DOX and ICG in LC60S-DI nanoparticles at different feeding ratios, showing efficient encapsulation of the theranostic agents. (f) Ultraviolet–visible absorbance of free DOX, free ICG, simple mixture of free DOX and ICG (DOX&ICG), and LC60S-DI nanoparticles. (g) DOX release from the LC60S-DI nanoparticles in phosphate-buffered saline (PBS) is pH dependent and can be precisely controlled with near infrared (NIR) laser irradiation. The arrows indicate laser irradiation at three different times. (h) Fluorescence intensity of free DOX solution and supernatant of LC60S-DI nanoparticle solution with the same DOX concentration after laser irradiation for 0, 1 and 3 min, further showing the drug release can be precisely controlled by dosing the NIR irradiation. (i) Pictures showing the colour change in the LC60S-DI (DOX/ICG=1:2) nanoparticles solution from green to yellowish after NIR laser irradiation due to the irradiation-induced release of DOX (red) from the nanoparticles. Before NIR irradiation, the red colour of DOX was quenched by the green colour of ICG. The unspecified ratios of DOX to LC60S nanoparticle and DOX to ICG for making the LC60S-DI nanoparticles were 1:20 and 1:1, respectively. All the NIR laser irradiation was for 1 min at 1.5 W cm⁻².

Figure 3. Controlled drug release in cells and enhanced anticancer capacity with high drug LC.

Viability of (a) PC-3 and (b) MDA-MB-231 cancer cells after treated with LC60S-DI nanoparticles at different concentrations without or with NIR laser (L) irradiation. (c) Confocal images showing the DOX and ICG delivered using LC60S-DI nanoparticles are mainly in the cytosol before NIR laser irradiation. With NIR laser irradiation, the enhanced delivery of DOX into the nuclei is evident probably due to the irradiation-induced release of DOX from the nanoparticles. The arrows indicate cell nuclei. Viability of (d) PC-3 and (e) MDA-MB-231 cancer cells after treated with LC60S-DI nanoparticles made at different drug feeding ratios (that is, different LCs) and NIR irradiation. The data show that the LC60S-DI nanoparticles with higher drug LC is more potent against both types of cancer cells at all the four doses. (f) The mean fluorescence intensity of DOX in PC-3 cells treated with LC60S-DI nanoparticles and laser irradiation showing a higher DOX LC results in significantly stronger DOX fluorescence in the cells. DOX (in LC60S-DI nanoparticles) concentration: 10 μg ml⁻¹. Error bars represent±s.d. (n>50). *P<0.05 (Mann–Whitney U-test). (g) Release of DOX from LC60S-DI nanoparticles in PBS showing that given the same DOX concentration (0.1 mg ml⁻¹), NIR laser irradiation can induce more drug release from the nanoparticles with a higher drug LC. The unspecified ratios of DOX to LC60S nanoparticle and DOX to ICG for making the LC60S-DI nanoparticles were 1:20 and 1:1, respectively. The NIR laser irradiation was at 1.5 W cm⁻² for either 3 (a–c) or 1 min (d–g).

Figure 4. Enhanced and controlled drug delivery to tumour.

(a) In vivo whole animal imaging of ICG fluorescence at different times after intravenous injection via the tail vein in the forms of free DOX&ICG, LC60S-DI nanoparticles and C60S-DI nanoparticles together with ex vivo imaging of ICG in tumour and five different organs collected after killing the mice at 28 h. The arrows indicate the locations of tumours in mice. (b) Ex vivo imaging of DOX fluorescence in the same tumour and organs. (c) Images and (d) the corresponding intensity of ICG fluorescence in blood drawn from the mice injected with LC60S-DI nanoparticles, C60S-DI nanoparticles and free ICG. The data indicate a significantly prolonged half-life of the LC60S-DI nanoparticles in blood circulation, compared with free ICG and C60S-DI nanoparticles. Error bars represent±s.d. (n=3). *P<0.05. (Kruskal–Wallis H test). (e) Fluorescence images of DOX in tumour of mice killed at 28 h post injection with saline, DOX&ICG, C60S-DI nanoparticles and LC60S-DI nanoparticles (with and without laser irradiation right before sacrificing the mice). The arrows indicate that for the treatment with LC60S-DI nanoparticles, NIR irradiation induces release of DOX from the nanoparticles so that it can enter the nuclei of cancer cells in tumour in vivo. Otherwise, the DOX stays with the nanoparticles in the cytosol with minimal cytotoxicity. The unspecified ratios of DOX to empty nanoparticle and DOX to ICG for making the DOX- and ICG-laden nanoparticles were 1:20 and 2:1, respectively. All the NIR laser irradiation was for 3 min at 0.7 W cm⁻².

Figure 5. Augmented safety and efficacy of cancer destruction in vivo.

(a) Photographs of mice on day 28 after nine different treatments. (b) Growth curves of tumours in the mice together with images of the tumours collected on day 28 after killing the mice. Error bars represent±s.d. (n=3 for C60S-DI+L group, n=6 for other groups). **P<0.01. (Kruskal–Wallis H test). (c) Weight of the tumours collected after killing the mice on day 28. Error bars represent±s.d. (n=3 for C60S-DI+L group, n=6 for other groups). **P<0.01. (Kruskal–Wallis H test). (d) Representative images of histology (H&E) of the tumours collected on day 28. (e) Body weight of the mice with the various treatments showing no significant difference between them. (f) Histological (H&E) images of major organs collected on day 28 showing no evident systemic toxicity of the LC60S nanoparticles. The unspecified ratios of DOX to empty nanoparticle and DOX to ICG for making the DOX- and/or ICG-laden nanoparticles were 1:20 and 1:1, respectively. All non-specified NIR laser (L) irradiation was for 3 min at 0.7 W cm⁻². The NIR irradiation was conducted at 12 h after intravenous administration of the various drug formulations.