Supramolecular Vesicles Based on Gold Nanorods for Precise Control of Gene Therapy and Deferred Photothermal Therapy

Open AccessCCS ChemistryRESEARCH ARTICLE1 May 2022Supramolecular Vesicles Based on Gold Nanorods for Precise Control of Gene Therapy and Deferred Photothermal Therapy Ludan Yue, Kuikun Yang, Jianwen Wei, Mengze Xu, Chen Sun, Yuanfu Ding, Zhen Yuan, Shu Wang and Ruibing Wang Ludan Yue State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Taipa, Macau SAR 999078 Google Scholar More articles by this author , Kuikun Yang State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Taipa, Macau SAR 999078 Google Scholar More articles by this author , Jianwen Wei State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Taipa, Macau SAR 999078 Beijing National Laboratory for Molecular Sciences, Key Laboratory of Organic Solids, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190 Google Scholar More articles by this author , Mengze Xu Cancer Center, Faculty of Health Sciences and Center for Cognitive and Brain Sciences, University of Macau, Taipa, Macau SAR 999078 Google Scholar More articles by this author , Chen Sun State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Taipa, Macau SAR 999078 Google Scholar More articles by this author , Yuanfu Ding State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Taipa, Macau SAR 999078 Google Scholar More articles by this author , Zhen Yuan Cancer Center, Faculty of Health Sciences and Center for Cognitive and Brain Sciences, University of Macau, Taipa, Macau SAR 999078 Google Scholar More articles by this author , Shu Wang *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] Beijing National Laboratory for Molecular Sciences, Key Laboratory of Organic Solids, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190 Google Scholar More articles by this author and Ruibing Wang *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Taipa, Macau SAR 999078 Google Scholar More articles by this author https://doi.org/10.31635/ccschem.021.202101029 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail In spite of being a promising therapeutic modality, gene therapy has limited clinical applications, mostly due to the lack of spatiotemporal resolution and inadequate efficacy. Herein, we present a facile strategy to remotely control intracellular gene expression by using gold nanorod- (Au NR) derived, host–guest interaction-mediated supramolecular vesicles as a gene carrier and photothermal transducer. Upon pulsed laser irradiation, mild photothermal conditions dissociate supramolecular vesicles to release gene and simultaneously activate heat shock protein-70 promoter (Hsp70) for spatiotemporally initiating gene expression inside cancer cells. Subsequently, upon introducing a polymeric guest species specifically into cancer cells, the dissociated Au NRs functionalized with macrocyclic host molecules could re-aggregate rapidly in cells to retard exocytosis of these NRs, thereby allowing deferred photothermal therapy to enhance the overall therapeutic outcome. Download figure Download PowerPoint Introduction Gene therapy has attracted increasing attention during the past decade as a promising treatment modality.1–3 However, the lack of temporal and spatial control in gene expression inevitably leads to non-specific toxicities in vivo. Although tissue- or organ-specific promoters have been encoded in therapeutic genes to improve specificity,4 this approach often involves a tedious fabrication process, and the encoded gene cannot be extended to other tissues or organs. Recently, Wang and co-workers5 developed photothermally responsive conjugated polymer nanoparticles (CPNs) to remotely control intracellular gene expression under external near-infrared (NIR) laser irradiation. This design could ensure tumor-specific gene activation and avoid non-specific organ toxicity. However, gene therapy alone often exhibits limited therapeutic efficacy for cancer due to the need of live cells to express genes, the viability of which is partly compromised during the therapeutic process. Therefore, gene therapy is often combined with other therapeutic modalities including photothermal therapy (PTT).6–8 In contrast, the excellent biocompatibility and photothermal properties of plasmonic nanomaterials have been leveraged in materials assembly for biomedical applications.9,10 For instance, Nie et al.11 and Chen et al.12 respectively developed gold nanomaterials-based vesicles for PTT and theranostics of cancers. However, upon intracellular disassembly with laser-induced heat, these Au nanospheres and nanorods (NRs) were cleared quickly via fast exocytosis and excretion,9–12 making deferred or secondary PTT nearly impossible. Although later administration of another batch of plasmonic vesicles (NVs) or Au NRs would allow secondary PTT for potentially synergistic therapy, this approach would introduce additional inorganic material burden, causing potentially higher toxicity. As a natural polysaccharide, hyaluronic acid (HA) possesses desirable biocompatibility, nontoxicity, and biodegradability. More importantly, HA accumulates at tumor sites and actively targets some tumor cells via CD44 receptors.13–16 Moreover, in our previous work,16 cucurbit[7]uril (CB[7]) grafted HA could induce supramolecular aggregation and fusion of mitochondria via strong host–guest interactions between the CB[7] moiety of CB[7]-HA and adamantane (Ada) residing on the surface of mitochondria, suggesting the potential of HA as a glue to induce molecular aggregations. Previously, CB[7]-tethered Au NRs (CB[7]-AuNRs) were developed to allow modular surface modification of the nanomaterials and PTT of cancer.17 Herein CB[7]-AuNRs were further leveraged as building blocks for self-assembly into Au NVs via host–guest interactions between CB[7]-AuNRs and adamantane (Ada)-terminated polymers. The resultant NVs were utilized to spatiotemporally control gene release and expression in cancer cells via NIR laser irradiation as a means of photothermal activation, due to the incorporation of heat shock protein-70 promoter (Hsp70).18 After the primary gene therapy, the disassembled Au NRs could be re-aggregated intracellularly upon addition of Ada-grafted HA,19,20 leading to extended intracellular retention and deferred PTT as the secondary treatment modality for promoted cancer therapy (Scheme 1). Scheme 1 | Au NVs for remote control of gene expression, and subsequent supramolecular re-aggregation of Au NRs to retard exocytosis for PTT. Download figure Download PowerPoint Experimental Methods Materials and characterization Tetrachloroauric acid tetrahydrate (HAuCl4·4H2O), L-ascorbic acid, silver nitrate (AgNO3), sodium borohydride (NaBH4), ethanol, 25% glutaraldehyde, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Ada, sodium hyaluronate (HA, MW of 50 K), 1-ethyl-3,3′-dimethyl-(aminopropyl)-carbodiimide (EDC) and N-hydroxy succinimide (NHS) were purchased from Aladdin and used as received. Cetyltrimethylammonium bromide (CTAB) was purchased from Sigma-Aldrich (St. Louis, MO). The transfection agent PolyjetTM was purchased from SignaGen (Beijing, China). A Milli-Q Integral system (Merck) was used to supply Milli-Q water. The morphology of NPs was observed by transmission electron microscopy (TEM; JEOL 2100F, Tokyo, Japan). The zeta potential of nanomaterials was measured on a Zetasizer (Malvern) at 200 kV. NMR spectra were obtained using a Bruker Ultra Shield 600 PLUS NMR spectrometer. Fluorescence spectra were obtained on a Thermo Scientific Lumina fluorescence spectrometer . Fluorescence images were acquired by confocal laser scanning microscopy (CLSM; Leica TCS SP8 MP, Germany) and inverted fluorescence microscope (Olympus IX73). Fourier transform infrared (FT-IR) spectra and UV–vis–NIR absorbance spectra were measured on Bruker IFS-66V/S and Shimadzu UV-1800 spectrophotometers. MTT assays were measured using a microplate reader (Infinite F200 Pro, TECAN). Intracellular uptake and cell apoptosis were both quantified by a flow cytometer (Beckman Coulter). Preparation of Au NRs Au NRs were prepared by following a seedless method.21 Briefly, the seed solution was formed by mixing CTAB (0.20 M, 50 mL) and HAuCl4·4H2O (1 mM, 50.0 mL) and vigorously stirring for 15 min. AgNO3 (4 mM, 3 mL) was added before rapidly adding 120 μL of HCl (37%) to the solution to obtain a pH of ∼11. Subsequently, ascorbic acid (85.8 mM, 750 μL) was added to the mixed solution and stirred until the solution became clear. NaBH4 (0.01 M, 75 μL) was rapidly added dropwise into the solution. The solution was kept for 5 h to grow, and the obtained Au NRs were purified three times by centrifugation (9000g, 30 min). Preparation of CB[7]-Au NRs Mercaptoundecanoic acid was dispersed in ethanol and added dropwise into the aqueous solution of Au NRs.17 The mixture was sonicated for 1 h before being vigorously stirred for another 24 h. Au-COOH NRs were collected by washing with ethanol three times. The (allyloxy)1CB[7] was prepared according to the previous report.22 The CB[7]-NH2 was obtained by a thiol–ene “click” reaction between (allyloxy)1CB[7] and cysteamine and confirmed by NMR. EDC, NHS, and Au-COOH NRs were dissolved in dimethyl sulfoxide (DMSO) and stirred for 3 h before adding CB[7]-NH2. The mixture was magnetically stirred for 24 h at room temperature. The acquired nanocomposites were collected by centrifugation and dispersed in water. The amount of CB[7] on the surface was calculated by a competitive fluorescence assay. Briefly, an excess amount of a fluorescent guest molecule acridine orange (AO) was used to bind with the free CB[7] to form CB[7]·AO. A known quantity of CB[7]-Au NRs was mixed with an excess amount (amount A) of Ada in water and shaken for 5 min before ultracentrifugation. Ada (amount B) remaining in the supernatant was measured by adding CB[7]·AO and the fluorescence was compared to the CB[7]·AO standard curve. A negative B value was the amount of CB[7]. Preparation of [email protected]/PEG NRs Polylactic acid (PLA) was prepared by a ring-opening polymerization (ROP) reaction of L-lactide in ethanol. The obtained PLA was used for further reaction with excess carboxyl Ada (Ada-COOH) catalyzed by DMAP and EDC in CH2Cl2 for 48 h. The excess Ada-COOH was removed by dialysis. The product was precipitated and washed with diethyl ether and then dried under vacuum. The obtained Ada-PLA was confirmed by 1H NMR ( Supporting Information Figure S1). Ada conjugated PEG (PEG-Ada) was synthesized by the reaction of PEG-NHS (MW = 5000) with excess Ada.23 Triethylamine (TEA) (105 mg, 1.0 mmol, 5.1 equiv) and mPEG-NHS (1 g, 0.2 mmol, 1.0 equiv) in 10 mL CH2Cl2 was sequentially added to a solution of Ada-COOH (187.7 mg, 1.0 mmol, 5.0 equiv), and the mixture was stirred at room temperature for 2 h. The solvent was subsequently removed in vacuo, and water was added to the reaction residue. The unreacted Ada was removed by centrifugation. The solution was dialyzed with dialysis cassette (MWCO, 2 kD) against water overnight and lyophilized to obtain Ada-PEG, which was confirmed by 1H NMR ( Supporting Information Figure S2). Excess Ada-PLA and Ada-PEG with the molar ratio of 1:1 was dissolved in dimethylformamide (DMF) respectively and dropwise added into CB[7]-Au aqueous solution. The mixture was stirred for 12 h and washed in DMF by centrifugation. Preparation of Au NVs [email protected]/PEG NRs as well as the plasmid were dispersed in 200 μL of chloroform and dropwise added into 1 mL 1% polyvinyl alcohol (PVA, MW 9000) aqueous solution under sonication at room temperature to produce a microemulsion. The plasmid loaded vesicles were obtained after 15 min sonication followed by 24 h stirring to evaporate the chloroform. Excess PVA was removed by washing with water. The vesicles without plasmid were prepared via the same method by dispersing [email protected]/PEG NRs in chloroform and dropwise adding to water to produce the microemulsion. Photothermal properties of Au NVs For measuring the photothermal performance of Au NVs, 1 mL aqueous dispersion of NVs (200 μg Au mL−1) was placed in a quartz cuvette and irradiated with an NIR laser (808 nm, 2 W cm−2, 300 s). A thermocouple probe with an accuracy of 1 °C was inserted into the solution to monitor the real-time temperature; the temperature was recorded every 10 s. The photothermal performance of Au NRs was determined under the same conditions as those for Au NVs. Quantification of plasmid concentration The plasmid concentration in Au NVs was quantified by measuring the P content by inductively coupled plasma mass spectrometry (ICP-MS). First, the plasmid loaded Au NVs were washed by water and dispersed in 100 μL water. Then, 100 μL aqua regia was added to decompose the Au and destroy the DNA structure. The solution was diluted by 1% HNO3 and the P content was measured by ICP-MS. The plasmid concentration was calculated per a standard curve ( Supporting Information Figure S5, Y = 99.527X − 1.6994). Release profile of Au NVs Release profile studies of Au NVs were conducted using Paclitaxel (PTX) as a model payload in a dialysis method. Phosphate-buffered saline (PBS) solution (pH 5.8, 15 mL) at 25, 42, and 37 °C, respectively, was used as the release medium to study the PTX release kinetics. The dialysate (100 μL) was removed and replenished at 0, 0.5, 1, 2, 4, 8, 12, 20, 24, 36, 40, 48, and 72 h. The PTX release was quantified by high-performance liquid chromatography (HPLC). The chromatographic conditions were as follows: the column used was an XDB C18 (4.6 × 250 mm, 5 mm), and the mobile phase consisted of acetonitrile and water (60/40, v/v). For the plasmid release profile study, Hsp70::EGFP plasmid was loaded into Au NVs and quantified via ICP-MS analysis of P according to the standard curve in Supporting Information Figure S5. Plasmid loaded Au NVs (at a plasmid concentration of 7.24 μg mL−1) were dispersed in 500 μL aqueous solution (pH 5.8, adjusted by HCl) and incubated at 25, 42, and 37 °C, respectively. The solution were centrifuged (1000 rpm, 3 min) at 0.5, 1, 2, 4, 8, 12, 20, and 24 h, respectively. The supernatant were collected at each time point and processed by aqua regia and diluted by 1% HNO3 for P content analysis by ICP-MS to evaluate the plasmid release profile. Cell culture and in vitro cytotoxicity assays B16 cell line (melanoma cell line) was cultured in 25 cm2 flasks using Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillin & streptomycin (PS) solution at 37 °C with 5% CO2. The biocompatibility of NVs was assessed by MTT assays using B16 cell lines (1 × 104 cells per well). Controlled EGFP expression by Hsp70:EGFP loaded Au NVs B16 cells were incubated with Hsp70:EGFP plasmid loaded Au NVs for 3 h. Subsequently, an 808 nm NIR laser (2 W cm−2) was used to irradiate cells for 90 s, and the laser was cycled between off and on for 10 s, for 45 cycles (totaling 15 min). The cells were further cultured under 37 °C for 24 h. A parallel group without NIR laser irradiation was used for comparison. A commercial transfection agent, PolyjetTM, was used for positive control of Hsp70::EGFP gene transfection. Cells were cultured by the polyplex for 3 h, and then HSP promoter was activated by heating the cells in a 42 °C incubator for 15 min. Cells were further cultured under 37 °C for 24 h. A parallel group without heating was used as a blank control. The internalized fluorescence images of the cells after washing with PBS were acquired by a CLSM. Quantitative PCR for p53 gene expression levels The mRNA was extracted by a kit from Beyotime (Shanghai, China). RNA (1 μg) was reverse-transcribed using the TaKaRa primeScript Reagent Kit. qPCR analysis was performed using TaKaRa SYBR Premix EX TaqTM II Kit with the primers. Values were obtained by the ViiA 7 Real-Time PCR system. DNA electrophoresis gel The PCR product was further analyzed by DNA electrophoresis gel. The mixture of p53 DNA (5 μL) and loading buffer (1 μL) was loaded onto a 2.5% (w/v) agarose gel containing 0.01% (v/v) GelRed, and then was subjected to electrophoresis in 0.5% TAE buffer at a constant voltage of 90 V for 15 min. Finally, the DNA bands were visualized using a UV Gel Image System. Biosafety evaluation B16 cells were incubated with Au NVs (Au concentration of 50, 100, 150, 200, and 250 μg mL−1) for 3 h, followed by pulsed NIR laser irradiation (808 nm, 2 W cm−2, 90 s laser on, 10 s laser off and 10 s laser on, repeated for 45 cycles) and subsequent incubation for 24 and 48 h, respectively. The cell viability was evaluated by MTT assays. A parallel group without NIR irradiation was conducted as a control. Cytotoxicity evaluation B16 cells were incubated with plasmid loaded Au NVs (Au concentration of 50, 100, 150, 200, and 250 μg mL−1, and plasmid concentration of 0.435, 0.937, 1.441, 1.945, and 2.447 μg mL−1) for 3 h, followed by pulsed NIR laser irradiation (808 nm, 2 W cm−2, 90 s laser on, 10 s laser off and 10 s laser on, repeated for 45 cycles) and further incubation for 24 and 48 h. The cell viability was evaluated by MTT assays. A parallel group without NIR irradiation was used as a negative control. A group in which the Hsp70::tp53 plasmid was transfected with the assistance of PolyjetTM into cells with and without heating at 42 °C for 15 min, followed by an equal incubation time, was conducted as positive controls. Flow cytometry Cells after incubation were processed by an Annexin V-FITC Kit. The fluorescence signal of Annexin V-FITC and PI (positive and negative) obtained by flow cytometer reflected the apoptosis rate. Preparation of HA-Ada Carboxyl Ada was reacted with N-Boc-ethylenediamine hydrochloride in DMF for 12 h in the presence of EDC and NHS. The N-Boc group was then activated in DMF before amidation with HA in the presence of EDC and NHS in anhydrous DMSO for 24 h. The obtained HA-Ada was then dialyzed in Milli-Q water before freeze-drying. The product was confirmed by 1H NMR ( Supporting Information Figure S8). HA-induced re-aggregation Cells were treated with plasmid loaded Au NVs for 3 h, followed by pulsed NIR laser irradiation (808 nm, 2 W cm−2, 90 s laser on, 10 s laser off and 10 s laser on, repeated for 45 cycles) and further incubation for 24 h. Excess HA-Ada (0.5 mg mL−1) was added to cells and co-cultured with cells for another 4 h. Cells were continuously irradiated for 10 min with an 808 nm NIR laser at the power density of 2 W cm−2. Cells were cultured for an additional 24 or 48 h before MTT assays. For Au content studies, cells were collected and sonicated for 15 min before further processing by aqua regia. The solution was diluted by 1% HNO3 and the Au content was measured by ICP-MS. Cytotoxicity of time-delayed PTT For cytotoxicity of time-delayed PTT studies in the absence of therapeutic gene payload, B16 cells were incubated with Au NVs (without any payload) for 3 h, followed by pulsed NIR laser irradiation (808 nm, 2 W cm−2, 90 s laser on, 10 s laser off and 10 s laser on, repeated for 45 cycles) and further incubated for 24 h. Excess HA-Ada (0.5 mg/mL) was added to cells and co-cultured for another 4 h. Cells were continuously irradiated for 10 min with an 808 nm NIR laser at the power density of 2 W cm−2. Cells were cultured for an additional 24 h before MTT assays. Safety of aggregated Au NRs For safety evaluation of aggregated Au NRs, 0.5 mg mL−1 HA-Ada was added into CB[7]-Au NRs colloidal solution to induce aggregation in the aqueous solution. The Au content was quantified by ICP-MS. B16 cells were incubated with HA-aggregated Au NRs at Au concentration of 50, 100, 150, 200, and 250 μg mL−1 for 4 h to afford the same uptake time with that in Au NVs re-aggregation studies. Excessive Au NRs were washed, and the cells were incubated for another 24 h before MTT assays. Ethics statement All animal studies were conducted in strict accordance with the Guide for the Care and Use of Laboratory Animals, and the procedures were approved by the Animal Ethics Committee, the University of Macau. In vivo computed tomography imaging experiments B16 tumor-bearing C57BL6 mice were intravenously (i.v.) injected with 200 μL Au NVs at 2.5 mg mL−1 Au concentration, and the mice were imaged pre-injection and 1, 2, 3, 4, 6, and 8 h after injection. The computed tomography (CT) signals in the tumor region were measured. The signal peaked at 6 h postinjection, thus 6 h was selected as the irradiation time point. Another group of B16 tumor-bearing C57BL6 mice were i.v. injected with 200 μL Au NVs at 2.5 mg mL−1 Au concentration, and the mice were imaged pre-injection and 1, 2, 3, 4, 6, 8, 12, and 24 h postinjection. The tumors of the mice were irradiated with pulsed NIR laser for 15 min (808 nm, 2 W cm−2, 90 s laser on, 10 s laser off and 10 s laser on, repeated for 45 cycles) at 6 h after injection. Moreover, HA-Ada (100 μL, 5 mg mL−1) was injected into the tumor at 24 h after Au NVs injection. The mice were further imaged at 2, 4, 6, and 12 h after HA-Ada injection. The CT signals in the tumor region were obtained and normalized over time. Tumor inhibition studies Thirty B16 tumor-bearing C57BL6 mice were randomly divided into six groups and given 100 μL of the following treatments: (1) PBS, (2) Au NVs, (3) Hsp70::tp53 plasmid loaded Au NVs, (4) pulsed NIR irradiated Hsp70::tp53 plasmid loaded Au NVs, (5) pulsed NIR irradiated Hsp70::tp53 plasmid loaded Au NVs with HA re-aggregation and continuous NIR irradiation, and (6) pulsed NIR irradiated empty Au NVs with HA re-aggregation and continuous NIR irradiation (at Au concentration of 2 mg mL−1, plasmid concentration of 20.064 μg mL−1 and HA-Ada concentration of 5 mg mL−1). The treatment was repeated at day 0, 3, and 6 three times. The tumor length (a) and width (b) were measured every two days by vernier caliper, and the tumor volume (V) was evaluated by following the equation: V = ½ ab2. The body weight of the mice in each group was measured and the survival rate of the mice was recorded every two days in the 14 day treatment. The mice were sacrificed and the tumors in each group were excised and weighed on day 14. The main organs and the tumor in each group were sliced and stained according to the p53 immunofluorescence, hematoxylin and eosin (H&E), and terminal-deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays. Results and Discussion As shown in Figure 1a, supramolecular NVs were prepared by self-assembly of amphiphilic Au NRs (Figure 1b) via CB[7]-Ada host–guest mediated interactions with an aspect ratio of 4:1, a CB[7] surface cap,17 and hydrophobic Ada-PLA ( Supporting Information Figure S1) and hydrophilic Ada-PEG ( Supporting Information Figure S2) anchors.24 The nanovesicles were 353 ± 119 nm in size (Figures 1c–1e). As shown by the FT-IR spectra in Supporting Information Figure S3a, the peaks at 1100 cm−1 are attributed to the –C–O–C– group of Ada-PLA and Ada-PEG, and the peaks at 1750 cm−1 are attributed to the C=O vibrations in Ada-PLA and (allyloxy)1CB[7], which are also observed in the spectra of Au NVs. The peak at 2850 cm−1, corresponding to –CH2– groups in Ada-PEG, is observed in the spectra of Au NVs, indicating the chemical structures of the Au NVs components. As shown in Supporting Information Figure S3b, the surface zeta potential of Au NRs is 36.97 mV because of the positively charged CTAB on Au NRs. CB[7]-Au NRs shows a modestly lower potential (31 mV) due to the presence of the negative-dipole C=O groups of CB[7]. Moreover, the surface zeta potential decreased dramatically to 2.35 mV after self-assembly in the presence of PLA and PEG. Meanwhile, the two characteristic localized surface plasmon resonance (LSPR) bands of Au NRs at 520 and 850 nm25 exhibited bathochromic shifts upon formation of Au NVs (Figure 1f) because of the plasmonic coupling effect of Au NRs in the shell of vesicles.26 The temperature of Au NRs and NVs aqueous solutions increased dramatically to 79 °C and 85 °C, respectively, under NIR irradiation for 5 min (Figure 1g) for several cycles. The moderately higher temperature in Au NVs solution than that in Au NR solution was also attributed to plasmonic coupling inside NVs.27 Figure 1 | Characterizations of Au NVs. (a) Fabrication of Au NVs. (b) TEM image of Au NRs. (c) TEM image of Au NVs (scale bar is 200 nm, inset scale bar is 100 nm). (d) Scanning electron microscopy (SEM) image (scale bar is 200 nm). (e) Dynamic light scattering (DLS) analysis of Au NVs. (f) UV–vis absorption of Au NRs and NVs. (g) Temperature evolution of water, Au NRs, and NVs (200 μg mL−1 Au) under laser irradiation (808 nm, 2 W cm−2) for 5 min, followed by cooling for 13 min, repeated for three cycles. Download figure Download PowerPoint Au NVs were expected to show thermal-responsive disassembly (Figure 2a), like previously reported Au NVs.9,10,28 The aqueous temperature of Au NVs was well-controlled in the range of 42–44 °C under pulsed 808 nm NIR laser irradiation (Figure 2b). This temperature range, lower than hyperthermia temperature (>45 °C),29 allows cells to survive ( Supporting Information Figure S4), making heat-inducible plasmid promoter activation possible to initiate downstream gene expression in live cells. Under this NIR irradiation condition, Au NVs rapidly disassembled into single Au NRs or small clusters of NRs (Figure 2c). Au NVs showed a low level of payload (PTX) leakage (only 16%) at 25 °C for 72 h, indicating good stability (Figure 2d). When incubated at 42 °C, approximately 63.85% and 84.35% of PTX was released in 24 and 72 h, respectively, in contrast to only 26.76% and 49.81% release in 24 and 72 h at 37 °C. Similar release profiles of plasmid were observed for plasmid loaded Au NVs at different temperatures measured via ICP-MS ( Supporting Information Figures S5 and S6). The significant payload release triggered at 42 °C would ensure precise control of plasmid release via pulsed NIR. Furthermore, Au NVs (with and without pulsed NIR irradiation) showed good biocompatibility with B16 cells ( Supporting Information Figure S7). Figure 2 | (a) Disassembly of Au NVs for payload release under NIR irradiation. (b) Temperature of Au NVs solution (Au concentration of 200 μg mL−1) under pulsed NIR laser irradiation (2 W cm−2): 90 s on followed by 10 s off, and subsequent 10 s on/off for 10 cycles. (c) Scanning electron microscopy (SEM) image of disassembled Au NVs after irradiation by pulsed NIR laser. Scale bar: 1 μm. (d) PTX release kinetics under 25, 37, and 42 °C, respectively. (e) HA-Ada induced re-aggregation of Au NRs. (f) Digital photo of Au NVs (1), Au NVs without (2), and with HA-Ada (3) post irradiation. (g) SEM image of re-aggregated Au NRs. Scale bar: 1 μm. Download figure Download PowerPoint Hypothetically, after the duration needed for photothermally-responsive gene therapy, PTT via NIR laser irradiation of Au NRs would eradicate the residual live tumor cells and enhance the overall therapeutic effects. However, fast exocytosis of Au NRs would not allow effective deferred PTT. In fact, CB[7] on the surface of NRs may be further leveraged to re-aggregate supramolecular Au NRs upon addition of a polymeric guest species that may bind with various Au NRs via strong host–guest interactions (Figure 2e) to retard the exocytosis of Au NRs. Thus, deferred PTT may become feasible. In this study, biocompatible and CD44-targeting natural polysaccharide HA (MW =50 K) was grafted with Ada (with a grafting rate of 19.83%, Supporting Information Figure S8), which may act as a glue to re-aggregate Au NRs (Figures 2f and 2g) via strong host–guest interactions between CB[7] and Ada. Figure 3 | Cellular uptake and promoter activation. (a) Fluorescence in B16 cells after incubation for 1, 2, 3, and 4 h with FITC-loaded Au NVs (sca