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Published in final edited form as: Biomaterials. 2019 Aug 29;223:119460. doi: 10.1016/j.biomaterials.2019.119460

Enzyme-induced in vivo assembly of gold nanoparticles for imaging-guided synergistic chemo-photothermal therapy of tumor

Kuikun Yang b,1, Yijing Liu c,1, Yin Wang d,1, Qilong Ren b, Hongyu Guo b, John B Matson d, Xiaoyuan Chen c,**, Zhihong Nie a,b,*
PMCID: PMC6764864  NIHMSID: NIHMS1539507  PMID: 31513993

Abstract

This article describes a nanoplatform based on matrix metalloproteinase (MMP)-responsive gold nanoparticles (AuNPs) for tumor-targeted photoacoustic (PA) imaging-guided photothermal therapy and drug delivery. AuNPs were grafted with complementary DNA strands, tethered with doxorubicin and coated with poly (ethylene glycol) via a thermal-labile linker and a MMP-cleavable peptide, respectively. The nanoprobes remained well-isolated in healthy tissues, but formed aggregates rapidly under MMP-abundant conditions. The DNA hybridization-induced assembly of the nanoprobes led to prolonged tumor retention and strong nearinfrared (NIR) absorption, which is beneficial to deep-tissue imaging and therapy. Compared with MMP-inert nanoprobes, our platform demonstrated significantly enhanced efficiency in PA imaging and photothermal conversion upon NIR irradiation. Meanwhile, doxorubicin could be released rapidly in response to the localized elevation of temperature, leading to synergistic chemo-photothermal therapy. The unique nanoplatform may find applications in effective disease control by delivering imaging and therapy to tumors with high specificity, safety, and universality.

1. Introduction

The integration of diagnostic and therapeutic functions into a single platform makes theranostics attractive for personalized cancer management. Gold nanoparticles (AuNPs) have been extensively studied as both photoacoustic (PA) contrast and photothermal (PT) therapeutic agents as well as delivery vehicles in cancer theranostics, due to their good biocompatibility, ease in synthesis and surface modification, strong light absorption, and efficient photothermal conversion [1–8]. However, spherical AuNPs do not exhibit strong near-infrared (NIR) absorption which is required for deep tissue penetration in PA imaging and PT therapy (PTT) [9]. Although larger AuNPs possess higher absorption in NIR, small AuNPs (10–100 nm) are typically more favored in cancer imaging and therapy, because of their prolonged circulation and enhanced tumor accumulation through the enhanced permeability and retention (EPR) effect [10–25]. Various gold nanostructures (e.g., gold nanorods, nanostars and nanocages) with relatively small sizes have been developed to show strong NIR absorption, but they may suffer from complex synthesis procedures or chemical contamination from cytotoxic surfactants such as cetyltrimethylammonium bromide [7,8,26–38].

The emergence of stimulus-responsive tumor targeting strategy brings new opportunities for the effective use of AuNPs in theranostics. In the stimulus-responsive therapies, assembly of small AuNPs into larger aggregates at the tumor site is induced by specific signals to facilitate their tumor accumulation due to the enhanced EPR effect and slower clearance of the larger assemblies. The absorption of AuNPs can be tuned toward NIR region via coupled surface plasmons in closely placed nanoparticles in assemblies. Various endogenous (e.g., acidic pH and up-regulated enzymes) and exogenous stimuli (e.g., light and temperature variation) have been exploited to induce assembly of AuNPs for efficient tumor theranostics, but the clinical translation of the activatable targeting strategy remains challenging due to i) insignificant red-shift of absorption after assembly of AuNPs; ii) slow assembly process (up to days); iii) shallow penetration depth of visible light as the stimulus; iv) nonspecific activation caused by some acidic materials or other external acid stimulation; and v) insufficient cellular internalization of AuNPs required for their intracellular assembly [39–45].

Here we reported an assembly-based tumor targeting strategy to increase the accumulation of doxorubicin (Dox)-tethered AuNPs in tumor tissues for imaging-guided combination therapy. Matrix metalloproteinases (MMPs), known to be overexpressed in many tumor types and present as extracellular or membrane-bound tumor markers [46–49], are exploited as the stimulus to induce extracellular assembly of Dox-tethered AuNPs in tumors. MMP-responsive AuNPs were designed and fabricated by attachment of complementary DNA strands on the nanoparticle surface, followed by attaching Dox onto AuNPs via a thermo labile 4′4-azobis(4-cyanovaleric acid) linker. PEG was coated onto Dox-tethered AuNPs with a peptide linker containing a substrate for MMPs, which not only improves the circulation half-life of the nanoparticles but also avoids their nonspecific assembly (Fig. 1) [50,51]. These nanoparticles remained monodisperse during circulation after intravenous (i.v.) injection due to their small size and PEG coatings. Upon reaching the MMP-rich tumor tissues, the shielding PEG layer was rapidly cleaved to expose the complementary DNA strands on nanoparticle surface. Driven by the DNA hybridization, the nanoparticles rapidly aggregated and accumulated in tumor extravascular interstitium and induced the redshift of their absorption, thus leading to enhanced imaging signals and photothermal efficacy of PA imaging and PTT under irradiation of an 808 nm laser. In response to the locally elevated temperature, the conjugated Dox could be released to enable an improved therapeutic efficacy in combination with PTT. Compared with conventional stimulus-responsive assembly strategies, our platform showed distinctive features beneficial for tumor theranostics: i) strong NIR absorption for PA imaging and PTT after assembly of AuNPs; ii) rapid assembly at tumor sites after the cleavage of PEG coatings by MMP; iii) extracellular assembly instead of intracellular assembly to ensure sufficient aggregation of AuNPs in tumors; and iv) enhanced tumor inhibition via the combination of NIR-induced hyperthermia and chemotherapy.

Fig. 1.

Fig. 1.

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Schematic illustration of MMP-induced aggregation of AuNPs in vivo for enhanced PAI/PTT of tumor.

2. Results

2.1. Design, synthesis and characterization of MMP-responsive nanoprobes

Citrate-capped AuNPs with a diameter of 38.9 ± 3.9 nm were synthesized and modified with complementary DNA strands on their surface (Fig. S1A). The DNA-modified AuNPs showed good stability in 0.3 M NaCl solution, while citrate-capped AuNPs rapidly aggregated (Fig. S1B). Dox was conjugated onto AuNPs via a thermal-labile linker (Dox-ACVA-PEG1000-SH). The hybridization between complementary DNA strands could induce the rapid assembly of Dox-tethered AuNPs (Dox-AuNPs) in phosphate-buffered saline (PBS, pH 7.4) (Figs. S1C–E). PEG was attached onto Dox-AuNPs to prevent the nonspecific aggregation. Only PEG with sufficiently large molecular weight (> 3.4 K) was found to effectively protect the Dox-AuNPs from assembly, as a result of the complete shielding of DNA strands by the hydrophilic PEG layer (Figs. S2A–C). Therefore, PEG5000-COOH was selected to conjugate with NH2-GPLGVRGC-SH and subsequently attached onto Dox- AuNPs via the C-terminus cysteine (PEG-pep-Dox-AuNPs, Figs. S3A and B). Successful attachment of Dox and PEG5000-GPLGVRGC-SH (PEG-pep-SH) onto AuNPs was verified by the increased hydrodynamic diameter of Dox-AuNPs and PEG-pep-Dox-AuNPs, respectively (Fig. S3C). The negative charge of AuNPs was significantly reduced due to the substitution of citrate by PEG-pep-Dox-AuNPs (Fig. S3D). PEG-pep-Dox-AuNPs were fabricated with varying weight ratio of Dox-ACVA-PEG1000-SH and AuNPs (from 1:1 to 12:1) to measure the loading content of Dox. For comparison, MMP-inert PEG5000-SH was also tethered onto Dox-AuNPs as control nanoprobes (PEG-Dox-AuNPs).

PEG-pep-Dox-AuNPs were well dispersed in both PBS buffer (pH 7.4) and DMEM supplemented with 10% fetal bovine serum (FBS) for more than 8 days without noticeable change in their UV–Vis absorption and hydrodynamic diameters, indicating the high physiological stability of AuNPs after coating of PEG-pep-SH (Figs. S3E and F). However, the hydrodynamic diameter of PEG-pep-Dox-AuNPs increased significantly from 73.2 ± 18.8 nm to 1.8 × 106 ± 1.4 × 105nm after incubation with MMP-2 for 120 min, accompanied by a clear red-shift in absorption from ~528 nm to the NIR region centered at ~724 nm, indicating the aggregation of AuNPs after MMP-induced cleavage of the PEG coating (Fig. 2A and B). The rapid assembly of PEG-pep-Dox-AuNPs was also verified by the time-dependent red-shift of UV–Vis absorption and size distribution of the nanoprobes in the presence of MMP, where a broad NIR absorption and a significant size increase of the nanoparticles were observed within 30 min incubation with MMP-2 (Figs. S4A and B). Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) further confirmed the assembly of PEG-pep-Dox-AuNPs in the presence of MMP-2. The monodisperse and well isolated nanoprobes transformed into large aggregates in the presence of MMP-2, due to the hybridization of complementary DNA strands-tethered Dox-AuNPs after detachment of the MMP-responsive PEG coating (Fig. 2C,D and Figs. S4C and D). In contrast, no noticeable agglomeration was observed after incubation of MMP-inert PEG-Dox-AuNPs with MMP-2, suggesting that the assembly of AuNPs is initiated specifically by the MMP-induced cleavage of the PEG coating (Figs. S4E and F).

Fig. 2. Characterization of MMP-responsive nanoprobes.

Fig. 2.

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(A) UV–Vis absorption and (B) corresponding hydrodynamic diameters of AuNPs, PEG-Dox-AuNPs with MMP-2, PEG-pep-Dox-AuNPs with and without MMP-2. (C, D) TEM images of PEG-pep-Dox-AuNPs after incubating for 120 min (C) without and (D) with MMP-2 (Inset bar: 200 nm). (E) Photothermal effect of various groups (50 μg ml−1) illuminated with 808 nm laser (1 Wcm−2). (F) Temperature profile of various groups after being irradiated with laser for 1 min and then switched off.

The strong and broad NIR absorption (650–850 nm) of the assemblies motivated us to assess the potential of these MMP-responsive PEG-pep-Dox-AuNPs in the PTT of cancer. Different groups of AuNPs were dispersed in ultrapure water and then irradiated with an 808 nm laser (1 W cm−2). The change in solution temperature was mapped and quantified by real-time thermal imaging using a thermal camera (Fig. 2E and F). No significant temperature change was observed in the control of ultrapure water. For groups of AuNPs, PEG-Dox-AuNPs with MMP-2 and PEG-pep-Dox-AuNPs without MMP-2 incubation, the solution temperature increased slightly by 6.3, 8.3 and 7.5 °C respectively, after irradiation at 808 nm (1 W cm−2) for 1 min, corresponding to the photothermal conversion efficiency (η) of 13.2%, 15.3% and 14.9%, respectively. In contrast, the NIR irradiation induced an increase in solution temperature by 24.4 °C within 1 min in the case of PEG-pep-Dox-AuNPs incubated with MMP-2 (η = 35.7%), indicating the efficient PTT after MMP-induced assembly of the AuNPs (50 rg mL−1).

2.2. In vitro assembly and cytotoxicity of PEG-pep-Dox-AuNPs

We evaluated the performance of PEG-pep-Dox-AuNPs for in vitro PTT and drug delivery using murine squamous cell carcinoma cells (SCC-7) as model cells due to their reported high MMP-2 expression [52]. The loading capacity (LDox) of Dox in PEG-pep-Dox-AuNPs could be tuned from 0.8% to 2.4% by controlling the weight ratio between Dox-ACVA-PEG1000-SH and AuNPs without compensation of the physiological stability of PEG-pep-Dox-AuNPs (Figs. S5A and B and Fig. S6A). Here we chose PEG-pep-Dox-AuNPs with LDox of 2.4% for further investigation and then studied whether MMP-induced aggregation of AuNPs affected the NIR triggered release of Dox from the nanocarriers (Fig. S6B). Control groups of PEG-Dox-AuNPs (MMP-2+ laser +), PEG-pep-Dox-AuNPs (MMP-2- laser +) and PEG-pep-Dox-AuNPs (MMP-2 + laser−) all demonstrated insignificant release of Dox (< 20%) after 3 h incubation. In contrast, 87.1% of the Dox conjugated on AuNPs was released from PEG-pep-Dox-AuNPs (MMP-2+ laser +) within 3 h, which is similar to the release of Dox from PEG-pep-Dox-AuNPs in 90 °C water bath (79.8%). The enhanced release of Dox originated from the efficient photothermal heating after the MMP-induced assembly of PEG-pep-Dox-AuNPs. The absorption redshift into the NIR region and the resultant localized photothermal heating upon 808 nm irradiation could cleave the thermal-labile linker between Dox and SH-PEG1000, thus releasing Dox rapidly from the aggregated nanoparticles. To verify the assembly of nanoparticles in the MMP-abundant extracellular matrix, SCC-7 cells were cultured for 12 h before being isolated from the culture media via centrifugation. The supernatant was supplemented with 10% fetal bovine serum (FBS) and then incubated with various groups of AuNPs for UV–Vis absorption measurement. Compared with AuNPs and PEG-Dox-AuNPs, PEG-pep-Dox-AuNPs showed a significant redshift in absorption to around 705 nm in SCC-7 culture media due to the MMP-induced assembly of AuNPs (Fig. 3A, Figs. S7A–C). However, the system did not exhibit redshift in absorption when the culture media was pretreated with GM6001, an inhibitor of MMP, suggesting that PEG-pep-Dox-AuNPs possesses an excellent specificity in response to MMP. The extracellular assembly of PEG-pep-Dox-AuNPs was also confirmed by the ICP-MS, where PEG-Dox-AuNPs showed 2.72-fold higher cellular internalization of Au than PEG-pep-Dox-AuNPs. This could be ascribed to the reduced cellular uptake of AuNPs after their assembly in the presence of extracellular MMPs (Fig. S8). The in vitro cytotoxicity of PEG-pep-Dox-AuNPs against SCC-7 cells in the presence and absence of laser irradiation and GM6001 was evaluated by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Fig. 3B). The viability of SCC-7 cells incubated with PEG-Dox-AuNPs and PEG-pep-AuNPs and irradiated with an 808 nm laser was also evaluated for comparison. No significant toxicity was found for cells treated with PEG-pep-Dox-AuNPs, NIR, PEG-Dox-AuNPs + NIR and PEG-pep-Dox-AuNPs + GM6001 + NIR at all the studied concentrations. In contrast, the PEG-pep-AuNPs + NIR and PEG-pep-Dox-AuNPs + NIR groups exhibited a dose-dependent cytotoxicity on the tumor cells with IC50 values of 28.6 and 12.9 μgmL−1 (Au concentration), respectively. The combination index (CI) was calculated to be 0.62 by using a Chou-Talalay method, indicating the significant synergistic effect between PTT and chemotherapy (Fig. S9) [53,54]. This suggests that the MMP-induced assembly of AuNPs is the prerequisite for efficient photothermal conversion and Dox release. The strong NIR absorption of nanoparticle aggregates in the presence of MMP-2 led to significantly enhanced heat production to eliminate tumor cells. Moreover, Dox could be rapidly released in response to the localized photothermal heating, leading to synergistic chemo-photothermal therapy. The live and dead staining analysis with Calcein AM (green) and ethidium homodimer (red) was consistent with the MTT results (Fig. 3C–H). No red fluorescence could be observed in the absence of NIR or MMP responsiveness, indicating the limited cytotoxicity of individual nanoparticles. However, PEG-pep-AuNPs showed significant inhibition of SCC-7 cells upon laser irradiation and the cytotoxicity could be further improved in the case of PEG-pep-Dox-AuNPs treated cells, indicating the superior synergistic effect of chemotherapy and PTT after aggregation of the nanoprobes.

Fig. 3. In vitro assembly and cytotoxicity of PEG-pep-Dox-AuNPs.

Fig. 3.

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(A) UV–Vis absorption of AuNPs, PEG-Dox-AuNPs with SCC-7 culture media, PEG-pep-Dox- AuNPs with SCC-7 culture media but with or without MMP-2 inhibitor (GM6001). (B) SCC-7 cell viability when incubated with the corresponding groups. (C-H) Fluorescence images of Calcein AM and Ethidium homodimer-1 co-stained SCC-7 cells for different groups: (C) PEG-pep-Dox-AuNPs only (50 μg ml−1), (D) NIR only (808 nm, 1Wcm−2), (E) PEG-Dox-AuNPs + NIR, (F) PEG-pep-Dox-AuNPs + MMP-2 inhibitor + NIR (G) PEG-pep-AuNPs + NIR and (H) PEG-pep-Dox-AuNPs + NIR (Scale bar: 200 μm).

2.3. In vivo distribution and tri-modality imaging

We evaluated the biodistribution and potential of PEG-pep-Dox-AuNPs as contrast agents for bimodal PA and PT imaging in vivo. PEG-pep-Dox-AuNPs were labelled with 64Cu and intravenously injected into SCC-7 tumor bearing mice (50 mg AuNPs/kg) to monitor the migration of the nanoparticles by positron emission tomography (PET) imaging (Fig. 4A and B). The 64Cu-labelled PEG-Dox-AuNPs were also fabricated and used as the MMP-inert nanoprobes for comparison. Compared with PEG-Dox-AuNPs, PEG-pep-Dox-AuNPs showed a higher tumor uptake at each time point. Quantitative analysis on the whole-body images showed the maximum tumor uptake of 5.59 ± 0.52 %ID/g of PEG-pep-Dox-AuNPs at 24h post-injection, which is 1.61-fold of PEG-Dox-AuNPs in tumor tissues (3.48 ± 0.18 %ID/g). The accumulation of PEG-pep-Dox-AuNPs was also confirmed by inductively coupled plasma mass spectrometry (ICP-MS) analysis, where 3.94 ± 2.01 %ID/g of PEG-pep-Dox-AuNPs were found in tumor at 24 h post-injection, which was 2.43-fold that of PEG-Dox-AuNPs (1.62 ± 0.13 %ID/g) (Fig. S10). The enhanced tumor accumulation of PEG-pep-Dox-AuNPs could be ascribed to the MMP-induced nanoparticle assembly and thereby reduced excretion rate and prolonged tumor retention (Fig. S11). The PA properties of the MMP-responsive nanoparticles were also evaluated with MMP-inert PEG-Dox-AuNPs as the control group. Many small granular areas with strong PA signals could be clearly observed in the PEG-pep-Dox-AuNPs treated tumor tissues (Fig. 4C). In contrast, only a few granular regions were distinguishable in tumors when PEG-Dox-AuNPs were administrated. The average tumor PA intensity of PEG-pep- Dox-AuNPs treated mice was 1.74-fold stronger than that observed from mice injected with PEG-Dox-AuNPs (Fig. 4D). A similar trend was observed in the thermal imaging of the tumor region after intravenous injection of various nanoprobes and irradiation with an 808 nm laser. The local tumor temperature rapidly increased by 17.8 °C within 10 min irradiation (1Wcm−2), which was sufficiently high to inhibit tumor growth in vivo. In comparison, the local temperature increase in tumors was only 8.6 and 8.8 °C for PBS and PEG-Dox-AuNPs treated mice, respectively (Fig. 4E and F). The enhanced PA imaging and PT efficacy of MMP-responsive nanoprobes could be ascribed to two aspects: 1) enhanced accumulation of nanoparticles in tumor tissues due to the MMP-induced assembly and 2) higher efficiency in photothermal conversion due to strong NIR absorption after assembly of nanoparticles.

Fig. 4. In vivo distribution and tri-modality imaging of PEG-pep-Dox-AuNPs.

Fig. 4.

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(A) Representative PET images of SCC-7 tumor-bearing mice at 1, 4, 24, 48 h postinjection (p.i.) of the 64Cu-labelled PEG-Dox-AuNPs (left) and PEG-pep-Dox-AuNPs (right). Tumors were circled with white dots, and (B) PET quantification of corresponding tumor uptake (n = 3, mean ± s.d.). (C) Representative PA images of SCC-7 tumors taken before and at different time points after intravenous injection of PEG-Dox-AuNPs (left) and PEG-pep-Dox-AuNPs (right). The maximum PA intensity was observed at 24 h p.i. in both groups, and (D) PA signal quantification in corresponding tumors (n = 3, mean ± s.d.). (E) Thermographic images of mice under 808 nm irradiation (1 W cm−2) at various time intervals 24 h p.i. of PBS, PEG-Dox-AuNPs and PEG-pep-Dox-AuNPs, and (F) corresponding curves of temperature rise at tumor regions during the irradiation.

2.4. In vivo synergistic chemo-photothermal therapy of tumor

We further assessed the antitumor efficacy of PEG-pep-Dox-AuNPs after systemic administration. The SCC-7 tumor-bearing mice were divided into seven groups and treated with: (a) PBS as the control group, (b) 808 nm laser irradiation, (c) free Dox (1.2 mg/kg), (d) PEG-pep-Dox-AuNPs (1.2 mg Dox/kg corresponding to 50 mg AuNPs/kg, same dose for the following groups), (e) PEG-Dox-AuNPs ( + laser), (f) PEG-pep-AuNPs (+ laser) and (g) PEG-pep-Dox-AuNPs (+ laser). The tumor volumes were measured every two days after laser irradiation on day 1 (Fig. 5A). It was found that the mice treated with PBS buffer exhibited a rapid increase in tumor size. Minor delay in tumor growth was observed in the mice treated with Dox, laser irradiation, PEG-pep-Dox-AuNPs or PEG-Dox-AuNPs (+ laser). Thanks to the enhanced photothermal efficiency, mice treated with PEG-pep-AuNPs (+ laser) exhibited improved efficacy in tumor growth inhibition. In contrast, the tumor growth was nearly completely inhibited for the mice treated with PEG-pep-Dox-AuNPs (+ laser), indicating the effective synergistic antitumor efficiency benefiting from the MMP-triggered assembly of the nanoprobes. Moreover, the mice treated with PEG-pep-Dox-AuNPs (+ laser) exhibited a much longer survival life without a single death or tumor recurrence (over 16 days) as compared to all the other groups (Fig. 5B). Notably, negligible loss of body weight was observed for all the groups of mice during the therapeutic period (Fig. 5C). Meanwhile, the histological analysis of the typical heart, liver, spleen, lung, and kidney of the mice treated with PEG-pep-Dox-AuNPs (+ laser) showed no significant damage to these organs, indicating the excellent biocompatibility of the MMP-responsive nanoprobes (Fig. 5D).

Fig. 5. In vivo synergistic chemo-photothermal therapy of tumor.

Fig. 5.

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(A) Tumor growth curve, (B) survival curve and (C) body weight variation of SCC-7 tumorbearing mice after different treatments: PBS, 808 nm laser (1Wcm−2), free Dox (1.2 mg/kg), PEG-pep-Dox-AuNPs, PEG-Dox-AuNPs + laser, PEG-pep-AuNPs + laser and PEG-pep-Dox-AuNPs + laser. Error bars from (A) to (C) represent the standard deviations of 4 mice per group. (D) H&E stained tissue sections after injection of PEG-pep-Dox-AuNPs + laser showing the non-toxicity of the nanotherapeutics (scale bar: 100 μm) *p < 0.05.

3. Conclusion

In summary, we developed an MMP-responsive nanoprobe for assembly-induced PA imaging and synergistic chemo-photothermal therapy of tumors. The nanoprobes remained stable with a stealth surface in blood circulation, but formed aggregates rapidly in extravascular tumor matrix where MMP is overexpressed than in normal tissues. An increase in size after assembly reduced the excretion rate of the nanoprobes thereby prolonged their tumor retention. Meanwhile, the strong NIR absorption after assembly made the nanoprobes attractive for PA imaging and PTT. Dox could be released rapidly from the nanoprobe aggregates under laser irradiation due to the photothermal effect and a significantly enhanced efficiency in both PA imaging and synergistic chemo-photothermal therapy was observed, as compared with the MMP-inert nanoprobes. This nanoplatform may find applications for tumor-targeted imaging and therapy and accelerate the clinical translation of enzyme-responsive nanoprobes for tumor theranostics.

Supplementary Material

1

Acknowledgment

Z. N. acknowledges the support of the Startup Fund from Fudan University and the Program for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning. X. C. is grateful for the support from the Intramural Research Program of the National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health. We also acknowledge the technical support from the Maryland NanoCenter and its AIMLab.

Footnotes

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.biomaterials.2019.119460.

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