Recent advances in matrix metalloproteinases-responsive nanoprobes for cancer diagnosis and therapy

2.1 MMP-1

MMP-1 belongs to the interstitial collagenase, and its substrates mainly include collagen types I, II, III, and V [24]. As a collagenase related with cancer, MMP-1 can promote tumor progress by degrading ECM [25]. Currently, upregulated MMP-1 expression has been found in a variety of cancer tissue specimens, and its high expression has been associated with poor prognosis of oral cancer, colorectal cancer, breast cancer, prostate cancer, and bladder cancer [26–30].

2.2 MMP-2/9

MMP-2 and MMP-9, both of which belong to gelatinases, play key roles in the physiological and pathological process of tumors [31]. On the one hand, they can specifically degrade the main structural components of ECM and basement membrane, such as gelatin and type IV collagen, which help tumor cells to infiltrate from the missing basement membrane into the surrounding tissues, thus promoting the invasion and metastasis of tumor cells [32,33]. On the other hand, studies have shown that MMP-2 and MMP-9 can promote the expression of vascular endothelial factors, which in turn reduce the formation of pannus, increase the permeability of the vascular wall, promote angiogenesis, and hence improve tumor growth [23,34,39]. MMP-2 and MMP-9, the most important MMPs, are abnormally expressed in various tumors, such as pancreatic cancer, breast cancer, bladder cancer, and cervix cancer [35,36].

2.3 MMP-7

MMP-7, also known as matrilysins, is the member of the matrix metalloprotease family with the smallest molecular weight [37]. MMP-7 is specifically expressed in various human tumors, including gastric cancer, pancreatic cancer, colorectal cancer, esophageal cancer, gallbladder cancer, bladder cancer, ovarian cancer, breast cancer, lung cancer, and other malignant tumors [36,38]. MMP-7 promotes tumor invasion and metastasis by inhibiting apoptosis, decreasing cell adhesion, and inducing angiogenesis [38].

2.4 MMP-14

MMP-14, also known as MT1-MMP, is a membrane-type MMP [40]. It has the widest substrate hydrolysis range, and the degradable zymogram involves type I collagen, type II collagen, fibronectin, laminin, hyaluronan, fibrin, proteoglycan, etc., [41]. Clinical studies have found that over-expressed MMP-14 is associated with poor prognosis in many kinds of cancer patients, such as melanoma, neuroblastoma, small cell lung cancer, pancreatic cancer, bladder cancer, breast cancer, and so on [42,43]. MMP14 is involved in the degradation and remodeling of ECM, promotion of tumor angiogenesis and regulation of cell adhesion, and affects the cellular microenvironment [42]. It is a key enzyme for tumor cell infiltration and migration. Studies have demonstrated that MMP-14 plays a role in the activation of pro-MMP2 and pro-MMP13, the upregulation of VEGF gene expression, and the mediation of CD44 shedding [44–47].

3.1 Fluorescence imaging (FI)

FI has shown considerable promise in applications of medical diagnosis, drug delivery, and image-guided surgery, which attributed to its benefits of non-invasive detection, high sensitivity, in situ operability, and high temporal resolution [57]. Owing to the high resolution and good biocompatibility of fluorescent probes, FI has become an attractive method for study of biomolecules, signal pathways, and biological reactions [58]. In recent years, a variety of fluorescent dyes have been developed, including organic dyes such as small molecule dyes, aggregation induced emission (AIE) nanoparticles, semiconductor polymer nanoparticles, and inorganic materials such as semiconductor quantum dots (QDs), metal nanoclusters, rare earth doped nanoparticles, and nanodiamonds [59,60]. At present, MMP-responsive fluorescent probes are mostly constructed based on fluorescence resonance energy transfer (FRET) principle to form an energy acceptor-polypeptide-energy donor system [61]. As shown in Figure 1, when the distance between energy donor and receptor is less than 10 nm, energy transfer occurs between the two, resulting in fluorescence quenching. When enzyme hydrolyzes the substrate polypeptide, the FRET system is damaged, thereby turning on the fluorescence and realizing detection of tissues [12,62].

Figure 1

FRET system based on enzyme response.

3.1.1 Organic fluorescent probes

Organic small molecule fluorescent reagents are a class of biological probes with excellent performance, featuring high sensitivity, good biocompatibility, and fast reaction time. Luan et al. developed Dab-GPLGVRGY-FITC fluorescent probes for rapid and accurate imaging of MMP-2 overexpressed gastric cancers [48]. Upon cleavage of GPLGVRGY by MMP-2, separation of Dab/FITC pairs decomposed the FRET system and activated fluorescence emission of FITC. Their results demonstrated that the nanoprobes possessed excellent sensitivity to MMP-2, which measured approximately 40-fold fluorescence enhancement at 520 nm with a detection limit of 42 ng·mL⁻¹. Xu et al. synthesized MMP-activatable I₇₈₀BP-PEG12 probes by labeling the MMP substrate peptide GPLGVRGKGG with near-infrared dye IR780 and quencher BHQ-3. Benefiting from the targetability of substrate peptides and long wavelengths of near-infrared fluorophores, the I₇₈₀BP-PEG12 probes exhibited highly sensitive detection of MMPs in tumor areas as low as nanomolar concentrations, which allowed deep tissue imaging and dynamic non-invasive evaluation. Their results showed that the I₇₈₀BP-PEG12 probes provided significant fluorescence enhancement in early stage of pancreatic ductal adenocarcinoma model with only 3 mm of tumor diameter [49].

In addition to fluorescence enhancement, the ratiometric fluorescent probes have unique advantages in detection of biological systems with high accuracy. The ratio type fluorescent probes show a change in enzyme activity by a proportional change in fluorescence intensity at two wavelengths [63]. Zeng et al. Designed ratiometric near-infrared fluorescent nanoprobes (SPN-MMP-RGD) for sensitive detection of MMP-2 activity in vitro [50]. As shown in Figure 2a, after self-assembly of semiconductor nanoparticles (SPNs) based on near-infrared absorption organic polymer (PCPDTBT), the nanoprobes were further modified with MMP-2 cleavable peptide GGPLGVRGK linked fluorophore Cy5.5 and its quencher QSY21, as well as labeled with cRGD ligand that could bind to tumor αvβ3 integrin. The results showed that upon interaction with MMP-2, fluorescence of SPN-MMP-RGD nanoprobes at 690 nm could activate fluorescence due to broken FRET structure between Cy5.5 and QSY21, while the fluorescence of PCPDTBT at 830 nm remained unchanged (Figure 2b), resulting in a significant enhancement of fluorescence ratio between 690 and 830 nm (I ₆₉₀/I ₈₃₀) by about 176-fold (Figure 2c).

Figure 2

(a) Scheme of preparation and activation of ratiometric NIR fluorescence of SPN-MMP-RGD toward MMP-2 under irradiation by 660 nm. (b) Fluorescence spectra and (c) fluorescence intensity ratio (I ₆₉₀/I ₈₃₀) of SPN-MMP-RGD (28 µg·mL⁻¹ PCPDTBT) before and after incubation with MMP-2 (8 nM). Reprinted with permission from Zeng et al. [50]. Copyright 2021, Wiley-VCH.

3.1.2 Inorganic fluorescent probes

Inorganic fluorescent nanomaterials have become a hotspot in the field of bioimaging due to their high fluorescence intensity, good photostability, and large Stokes shift, which include three major categories: QDs, rare earth fluorescent materials, and noble metal nanoclusters [64–66]. Gold nanoparticles (AuNPs) are widely used for FI owing to their good biocompatibility and high quenching efficiency. Guo et al. constructed a novel tricolor fluorescence nanoplatforms based on a high-fidelity gold-selenium (Au-Se) bond for simultaneously imaging and in situ monitoring the expression level of MMP-2/7/9 protein [51]. The nanoprobes were conjugated with three peptide substrates labeled with fluorescein isothiocyanate (FITC), 5-carboxytetramethylrhodamine (5-TAMRA), and cyanine 5 (Cy5), respectively. When hydrolyzed by MMP-2/7/9 in tumor, the previously quenched fluorescence by FRET was recovered, so that the biomarker MMP-2/7/9 could be visualized to elucidate the invasion and migration behaviors of tumor cells in an inflammatory environment. QDs are fluorescent nanocrystals with excellent characteristics for biological imaging, including adjustable spectral range, strong anti-photobleaching ability, and easy surface modification [67]. For instance, Chung et al. developed quantum dot-based fluorescence resonance energy transfer (QD-FRET) nanosensors to visualize the activity of MT1-MMP on cell membranes. Via FRET and penetrated QD signals, the nanosensors could profile cancer cells [52]. Pham-Nguyen et al. reported a tumor sensing system based on AuNPs and QDs via an MMP-cleavable linker. The fluorescence of the QDs was quenched by AuNPs based on FRET, allowing fluorescence monitoring of tumor-specific enzymes in vivo levels (Figure 3a) [53]. When AuNPs and QDs reached tumor with overexpression of MMP-2, the methoxy PEG as a protective layer was removed and the azide fraction was exposed, which clicked with QDs to form Au-QD nanoclusters. The fluorescence of QDs was quenched by AuNPs due to FRET effect. As shown in Figure 3b, fluorescence quenching of QDs occurred within 1 h after exposure to 0.25–1.0 nM of MMP-2. Furthermore, it was demonstrated that the FRET efficiency was proportional to the MMP-2 level (Figure 3c).

Figure 3

(a) Scheme of preparation and enzyme-responsive cleavage of nanoclusters for real-time imaging. (b) IVIS images and (c) FRET efficiency of Au-QD nanoclusters at various incubation times and MMP-2 concentrations. Reprinted with permission from Pham-Nguyen et al. [53]. Copyright 2022, American Chemical Society.

3.2 Multimodal imaging

FI has high sensitivity but is limited by tissue penetration depth, while magnetic resonance imaging (MRI) has high soft tissue resolution, unlimited tissue penetration, but low sensitivity. In addition, computed tomography has high spatial resolution but carries radiation risks [68]. Therefore, compared with single imaging, integrating the complementary advantages of multiple imaging modalities can provide more comprehensive and detailed information for precise diagnosis of tumors.

MRI contrast agents combined with photoacoustic imaging agents are expected to produce complementary imaging effects. Melanin has become a potential photoacoustic contrast agent due to its excellent photoacoustic properties and near-infrared absorption capability. Meng et al. chelated Gd³⁺ on melanin nanoparticles, making them possess dual-mode MRI/PA imaging ability [54]. In addition, due to modification of MMP-2 peptide substrate, the nanoparticles were hydrolyzed under MMP-2 enzyme to trigger exposure of the hydrophobic end, leading to nanoparticles accumulated in tumor with longer time of retention, finally showing an excellent imaging effect.

At present, dual-mode imaging strategies are commonly used including FI/MRI, PA/MRI, CT/MRI, and FI/SERS, etc. Among these strategies, the FI/SERS detection platform has proved to be a very reliable analytical tool in the field of biosensing. Taking advantage of the high sensitivity and fluorescence visualization of SERS, Liu and colleagues designed a novel hybrid nanosensor to accurately monitor MMP-2 activity in cell secretions and human serum samples [55]. Specifically, the prepared nanosensor (FMNS@Au) were based on biological self-assembly, in which MMP-2 peptide substrate (PLGVR) acts as a bridge between AuNP and fluorescent magnetic nanospheres (FMNS), forming a FRET system. Under the action of MMP-2, the FRET system was damaged, meanwhile the “hot spot” effect of SERS was weakened, which resulted in recovery of fluorescence signal and reduction in SERS signal, and thereby improving detection sensitivity of MMP-2.

Surgery is the main treatment of cancer, and the extent of surgical resection is closely related to the prognosis of patients. Accurate tumor resection, especially for the tumors located in complex position in body, such as glioma, is an urgent problem to be solved. Currently, multimodal imaging-guided surgery has become a hot research field. MMP-14 is overexpressed in glioma, which can be used as a biomarker for molecular imaging of glioma. Kasten et al. designed dual-mode imaging probes for MMP-14 targeting, and applied them in near-infrared fluorescence (NIRF)/PET-guided resection of glioma (Figure 4a) [56]. The probes consist of two peptide sequences, one is MMP14 substrate peptide with near-infrared fluorophore IRDye800 and the quencher QC-1-NHS attached to each end, and thus can be used for MMP-14 activatable NIRF imaging, and the other is peptide labeled with radioactive elements ⁶⁴Cu(ii) or ⁶⁸Ga(iii) for PET imaging (Figure 4b). As shown in Figure 4c, U87 and U251 cells showed high levels of MMP-14 expression by immunofluorescence, demonstrating the ability of these cell lines to detect enzyme substrates. This was also confirmed by the results of Figure 4d, where the NIRF signals of U87 and U251 cells were higher under the NIRF microscope when the substrate peptide and the substrate-binding peptide were incubated with the glioma cell line in vitro. In addition, Figure 4e–g illustrates that the MMP-14 probe, after intravenous injection in mice bearing in situ PDX JX12 glioma, shows superior NIRF/PET imaging capability with significant contrast at the tumor site relative to normal brain tissue. The results indicated that PET and NIRF signals were linearly related and co-localized with MMP-14 expression in resected tumors in orthotopic patient-derived xenograft glioma tumors.

Figure 4

(a) Scheme for dual-modality PET/NIRF imaging of glioma (GBM) with an MMP-14 activatable peptide. (b) Structure of MMP-14 substrate-binding peptide probe. (c) Immunofluorescence signal quantification of MMP-14 in GBM adherent cells cultured in vitro. (d) Cell-associated NIRF signals (red) of glioma cells (D54, U87, U251) after incubation with substrate binding peptide (top), substrate peptide (middle), or buffer control without peptide (bottom) for 1 h. (e) NIRF imaging of mouse tissue sections harboring in situ PDX JX12 glioma tumor 1 h after intravenous injection of substrate-binding peptide. (f) PET images in mice bearing in situ PDX JX12 glioma tumors 4 h after intravenous injection of ⁶⁴Cu substrate binding peptide. (g) In vitro biodistribution showing whole brain activity 5.5 h after intravenous injection of ⁶⁴Cu substrate binding peptide or ⁶⁴Cu substrate binding peptide + blockade (non-labeled binding peptide) in mice bearing in situ PDX JX12 glioma tumors. Reprinted with the permission from Kasten et al. [56]. Copyright 2019, Springer Nature.

4.1 Chemotherapy

Traditional chemotherapeutic drugs lack targeting, which will cause toxicity to normal tissues [16]. Taking advantage of specially overexpressed MMPs in tumor, targeted drug delivery system can accurately deliver and control release drugs in tumor site, thereby reducing side effects and improving the treatment effect [85]. Doxorubicin (DOX) is an anthracycline that has been used as first-line drug in clinical treatment of malignant tumors such as breast cancer, malignant lymphoma, and lung cancer [86]. Due to the concomitant toxic and side effects, such as cardiac toxicity and liver injury, DOX is seriously restricted in clinical treatment of cancer [87]. For improving pancreatic cancer treatment and reducing side effects of DOX, Wei et al. developed an intelligent response-type nanovesicle MC-T-DOX loaded with DOX (Figure 5) [72]. After intravenously injecting MC-T-DOX in pancreatic cancer model, MC-T-DOX was activated by MT1-MMP from tumor endothelial cells (ECs) to release selegiline, thereby promoting ECs migration and angiogenesis, and improving the accumulation and distribution of MC-T-DOX at tumor site. The DOX was then released under thermal triggered by MC-T-DOX, which increased bioavailability. Ryu et al. further improved DOX delivery system by carrying an MMP-2 responsive polypeptide linker, which showed better cell penetration and could induce more cancer cell death [73]. Paclitaxel (PTX) is another first-line chemotherapeutic drug. Duan et al. designed MMP-triggered liposomes that sequentially loaded quercetin and PTX for fibrotic tumor microenvironment (TME) remodeling and chemotherapy boosting [74]. After administration, the liposomes specifically accumulated in stroma-rich tumor sites, and the two drugs were released when undergoing MMP digestion.

Figure 5

Scheme of MC-T-DOX enhances tumor blood perfusion and drug delivery in pancreatic cancer. MT1-MMP, membrane type 1-matrix metalloproteinase; MC, MT1-MMP-activated cilengitide. Reprinted by permission from Wei et al. [72]. Copyright 2020, Wiley-VCH.

4.2 Phototherapy

Phototherapy, including photodynamic therapy (PDT) and photothermal therapy, is a non-invasive treatment with features of high safety and high selectivity [88]. PDT utilizes photosensitizers to produce cytotoxic reactive oxygen species under irradiation of near-infrared light to induce apoptosis [89]. However, most of the photosensitizers lack cancer targeting capability and tissue penetration. It has been clarified that MMPs-responsive probes can improve the penetration of photosensitizers and achieve PDT in deep tumor [90]. Based on this strategy, Liang et al. overcame physiological barriers caused by TME and delivered zinc phthalocyanine photosensitizers (ZnPc) into deep tumor, which enhanced therapeutic efficiency [75]. As shown in Figure 6a, the nanoprobes consisted of two parts: (1) ferritin nanocage (PFRT) encapsulated ZnPc and (2) dendritic mesoporous silicon nanoparticles (DMSN) loaded oxygen-supplied hemoglobin (Hb)/matrix remodeling reagent (iTGFb). The two parts were linked by MMP-2 substrate peptide to form core-satellite nanoframeworks (T-PFRT) with convertible dimensions. After the nanoframeworks were cleavaged into two parts by MMP-2, DMSN released Hb and iTGFb led to normalization of TEM, which further promoted deep tissue penetration of PFRT, and finally improved PDT effect of ZnPc. As shown in Figure 6b, compared with addition of enzyme inhibitor SB-3CT, DMSN- PFRT showed more deep penetration of released PFRT in tumor spheroids. The tumor size curve in Figure 6c indicates that laser irradiated T-PFRT showed superior tumor suppression over the other groups. Similarly, Fan et al. combined catalase mimetics (Cerium oxide, CeOx) and photosensitizers with MMP-2 peptide substrate to overcome tumor hypoxia in PDT [76]. When the peptide (EGPLGVRGK) was cut by MMP-2 at cleavage site between V and G, the smart nanoprobes changed from “silent state” before reaching cancer cell to “activated state” inside cell, thus turning on fluorescence and generating ¹O₂.

Figure 6

(a) The structure and composition of nanoframeworks T-PFRT. (b) Penetration depth of different formulations in 3D tumor spheroid. (c) Tumor growth curve in different groups. G1, PBS; G2, T-PFRT without laser irradiation; G3, DMSN-PFRT (w/o pep) with laser irradiation; G4, DMSN-PFRT with laser irradiation; G5, iTGFbDMSN-PFRT with laser irradiation; G6, OxyHbDMSN-PFRT with laser irradiation; G7, T-PFRT with laser irradiation. Reprinted with permission from Liang et al. [75]. Copyright 2021, Wiley-VCH.

Different from PDT, photothermal therapy (PTT) is a method that converts the absorbed near-infrared light into thermal energy by photothermal agent to ablate cancer cells [91].The principle of PTT is that cancer cells have lower heat tolerance than normal cells, and heat can cause irreversible damage to cancer cell membranes and trigger protein denaturation [92]. Although PTT has made great progress in cancer treatment, non-specific PTT reagents have certain limitations, such as inefficiency accumulation in tumor, low photothermal conversion, and inevitable damage to normal tissue [93,94]. In order to minimize the damage to non-pathological tissues, Wu et al. modified gold nanorods with MMP-9 sensitive peptide sequence (GPLG) [77]. Compared with nude gold nanorods, peptide modified gold nanorods showed enhanced accumulation in tumor, and therefore improved treatment efficiency.

4.3 Immunotherapy

Immunotherapy is a method of killing cancer cells by activating human autoimmune system [95]. Utilizing enzyme-specific hydrolysis to construct nanocarriers for immunotherapeutic drug delivery is a common strategy for MMPs-based immunotherapy. Liu et al. designed NF-κB pathway inhibitor IMD-0354 and programmed cell death protein 1 (PD-1) antibody co-loaded enzyme-responsive nanoparticles. Under decomposition by MMP-2, the nanoparticles released drugs could promote immune checkpoint-blocking immunotherapy by inducing polarization of tumor-associated macrophages and inhibiting the programmed cell death protein-1 (PD-1)/Programmed cell death 1-ligand 1 (PD-L1) pathway [78]. Similarly, Lu et al. exploited the abundant MMP-2 in TME to initiate tumor lysis and release tumor-associated antigen, addressing the problem of colorectal cancer insensitivity to anti-PD-L1 immunotherapy due to the lack of neoantigen [79]. In addition, D-peptide antagonist (DPPA-1), an immune checkpoint inhibitor, is often applied in immunotherapy as a polypeptide sequence with PD-1/PD-L1 blocking function due to its convenience in modification and strong penetration [80]. For example, Dai et al. used MMP-2 substrate peptide to immobilize immune checkpoint inhibitor ^DPPA-1 on outer membrane (Figure 7a). High expression of MMP-2 in tumor could reactively release ^DPPA-1 to block PD-1/PD-L1 pathway and activate anti-tumor immune response, thereby greatly inhibiting tumor growth and metastasis (Figure 7b). Compared with control group, tumor fluorescence intensity of the DIR@PLGA@MD group was significantly increased, which indicates excellent tumor targeting ability of the nanoprobes (Figure 7c). Figure 7d shows that AFT/2BP@PLGA@MD exhibited significant tumor inhibition (71.9%) and improved survival. In addition, AFT/2-BP@PLGA@MD significantly inhibited metastatic nodules in lung after 34 days (Figure 7e). Currently, coupling MMP-substrate peptides with ^DPPA-1 peptides to achieve responsive release in TME is a common strategy [81]. In conclusion, enzyme-mediated delivery system can improve drug efficacy, reduce immunotoxicity, and provide a platform for synergistic cancer immunotherapy.

Figure 7

(a) Scheme of preparation of AFT/2-BP@PLGA@MD nanoparticles. (b) The antitumor mechanism of AFT/2-BP@PLGA@MD. (c) FI of mice administration by various nanoparticles labeled with DIR. Fluorescence images of tumors and main organs of mice intravenously injected at 48 h and quantitative analysis of fluorescence intensity of ex vivo images. (d) Mice survival curves. (e) Images of metastatic nodules in lungs. Reprinted with permission from Wang et al. [80]. Copyright 2022, American Chemical Society.

4.4 Combination therapy

Despite the fact that individual medicines have anti-tumor effects, it is still necessary to construct multifunctional therapeutic platform to deal with the complex TME, and enhance therapeutic efficiency [96]. It has been demonstrated that combination of two or more therapeutic techniques can achieve more effective treatment and minimize incidence of tumor recurrence. Because of convenient modification, the MMPs substrate-based responsive strategy can integrate multiple drugs or treatment modalities to achieve multifunctional combination therapy.

As a representative, Hu et al. prepared enzyme-responsive MA-PEPA-Ce6 nanoparticles, on which PD-L1 inhibitor (metformin, MET) and photosensitizer (chlorin, CE6) were loaded via substrate peptide GPLGVRGDK [82]. After degraded by MMP-2 in tumor, the exposed VRGDK-Ce6 could specially bind with integrin αvβ3 receptor, and induce a strong anti-tumor immune effect under laser irradiation. Meanwhile, the released MET in acidic TME could further amplify the anti-tumor immune response, therefore combinedly inhibiting tumor growth. Self-assemble of amphiphilic peptides containing both hydrophilic and hydrophobic groups is another effective strategy for drug carriers. As shown in Figure 8a, Zhu et al. synthesized amphiphilic polypeptide by using MMP-2-sensitive peptide PLGLAG to graft hydrophobic radiotherapy sensitizers 2-(2-nitroimidazol1-yl) acetic acid (NIA) and hydrophilic PD-L1 antagonist ^DPPA-1 [83]. Further, as-synthesized amphiphilic peptides were co-assembled with hydrophobic immune adjuvant R848 to form nanoparticles. As shown in Figure 8b–d, MMP-2 responsive release of NIA, R848, and D1 peptide dramatically enhanced radiation sensitivity of tumor cells, promoted maturation of dendritic cells, and blocked PD-1/PD-L1 pathway, respectively. Therefore, both primary tumor and distal tumor were inhibited, and long-term immune protection was generated to prevent tumor recurrence.

Figure 8

(a) Antitumor mechanism of NIA-D1@R848. (b) Pictures of primary tumors and abscopal tumors after different treatments at day 23; (A–F) represent mice treated with saline, RT, R848, NIA-D1@R848, NIA-D1@R848 + RT, and C16-D1@R848 + RT at an equivalent R848 dose of 0.5 mg·kg⁻¹, respectively; scale bar = 1 cm. (c) Growth curve of primary and abscopal tumors. (d) Tumor images and growth curve of tumors from control naive mice and NIA-D1@R848 + RT cured mice. Scale bar = 1 cm. Reprinted with the permission from Zhu et al. [83]. Copyright 2022, Wiley-VCH.

For considerable enhancing treatment efficiency, three or more therapeutic medications have been successfully mounted on the same nanoplatform. Gao et al. designed novel nanoplatforms consisting of abemaciclib-loaded photothermal nanoparticles (A/Au@MSMs) and MMP-2 substrate peptide-conjugated anti-PD-1 antibody [84]. The nanoplatforms carried chemical-photothermal-immune synergistic therapy in one treatment, which completely eliminated tumor and showed great potential for cancer treatment in future.

5.1 Single mode image guided tumor therapy

MRI has been widely used in cancer diagnosis due to its advantages of non-invasive detection, high spatial resolution, and infinite tissue penetration depth [103]. Commonly used MRI contrast agents in clinic include T₁ contrast agents such as gadolinium-based (Gd³⁺) or manganese-based (Mn²⁺) small molecular, and T₂ contrast agents such as superparamagnetic iron oxide nanoparticles (SPIONs) [104,105]. The synergy of MRI contrast agents with cancer therapy show wide application prospect in tumor detection and drug release monitoring. Chen et al. designed novel MMP-9 responsive nanoplatform (PMP@USPIO/DOX, PMPSD) with T₂–T₁ conversion characteristics for tumor imaging and synergistic chemo-photothermal therapy [97]. Under physiological condition, the ultrasmall superparamagnetic iron oxide (USPIO) was in aggregated state and acted as T₂ contrast agent. However, when polypeptide chains were cleaved by MMP-9 in TME, the released USPIO transformed into T₁ contrast agent, which could obtain non-invasive imaging and quantitative analysis of MMP-9. In addition, the nanoplatform also shows excellent photothermal performance. Wang et al. synthesized multifunctional metal–organic frameworks modified with MMPs substrate peptide-conjugated bone targeting peptide and cell penetrating peptide for MRI guided chemo-photothermal therapy of bone tumor. The multifunctional metal-organic frameworks showed increased uptake in bone tumor cells, therefore enhancing photothermal-chemotherapeutic efficacy [98].

In addition to MRI, near-infrared FI-guided cancer therapy can supply high-sensitive optical signal of focus, thereby expecting to achieve precise treatment and real time prognosis evaluation. Chen et al. designed an image-guided drug delivery strategy using supramolecular AIE nanodots to achieve targeted drug release within tumor cells [99]. As shown in Figure 9a, the nanodots are composed of three parts, including two groups of functional alpha-cyclodextrin (alpha-CD) modified with anticancer drug gemcitabine (GEM) and AIE luminescent agent TPR, and a group of cell-penetrating peptide RRRRRRRRRR (R8) and zwitterionic stealth sequence EKEKEKEKEKEKEKEK (EK6) connected by MMP-2 peptide substrate PLGLAG. After MMP-2 degradation in tumor, the shielding sequence EK6 was removed to expose R8 to enhance intracellular internalization, and then release of GEM was triggered by intracellular reduction microenvironment. Compared to the control group, the nanodots showed enhanced fluorescence signaling and antitumor activity, demonstrating the targeting of MMP-2 in tumors (Figure 9b–e).

Figure 9

(a) Illustration of preparation of α-CD-TPR-GEM-mmp (+) NP. (b) Fluorescence images of main organs and tumors of mice after administration of nanoparticles for 24 h. (c) Quantitative fluorescent signals of main organs and tumors in (b). (d) In vivo bioluminescence images of orthotopic pancreatic tumor-bearing mice in different treatment groups. (e) Tumor growth curves through quantitatively analyzing the in vivo bioluminescence signals from pancreatic tumors in different treatment groups. Reprinted with permission from Chen et al. [99]. Copyright 2020, American Chemical Society.

5.2 Multimodal image-guided tumor therapy

Multimode imaging-guided therapy can overcome limitations of single-mode imaging and show higher application prospects. Shi et al. constructed a novel Gd³⁺ doped CuS magnetic SPNs (T-MAN), and then covalently modified with cRGD-targeted peptide, NIR fluorophore (Cy5.5), and quencher QSY21-labeled MMP-2 cleavable peptide substrate (Figure 10a) [100]. Following intravenous administration, T-MAN was actively delivered to gastric cancer tissues mediated by αvβ3 integrin and specifically cleaved by MMP-2 in ECM, which produced significantly enhanced NIR fluorescence and T₁-weighted MR contrast signals to accurately depict gastric tumors (Figure 10b). Under the guidance of NIR/MR bimodal imaging, in situ gastric tumors were ablated by 808 nm NIR laser. Figure 10c–e indicated that when interacting with MMP-2, T-MAN exhibited a large fluorescence turn-on ratio at 690 nm (∼185-fold), a high r1 relaxation value (∼60.0 mM⁻¹·s⁻¹), and preferential tumor accumulation (∼23.4% ID%/g at 12 h).

Figure 10

(a) Scheme of preparation of T-MAN nanodisks. (b) Fluorescence/MR imaging and PTT of in situ orthotopic gastric tumors. (c) Fluorescence spectra of T-MAN (0.5 μM MMP-2 substrate) incubated with MMP-2 (10 nM) for different times at 37°C. (d) r1 value of T-MAN and Dotarem at 1 T. (e) Distribution of T-MAN in MKN45 tumors and main organs of mice after administration. Reprinted with permission from Shi et al. [100]. Copyright 2019, American Chemical Society.

In addition to PTT, PDT-based diagnosis and treatment is also a research focus. For instance, Yang et al. modified amphiphilic DSPE-PEG2000 with MMP-2 substrate peptide PLGVRGRGDC and gadolinium chelating agent DTPA (diethylenetriamine pentaacetic acid), respectively, to form functionalized nanoparticles by self-assembly. Then, photosensitizers Ce6 and gadolinium ions were loaded on the nanoparticles. Their results showed MMP-triggered targeting peptides could specifically deliver Ce6 photosensitizers into A549 tumor cells and effectively ablate cancer cells under laser irradiation with guidance of NIRF/MR dual-mode imaging [101]. Furthermore, by using MMP-2/9 sensitive peptide as linker, Liu et al. prepared ultra-small gold nanoparticles with functions of three-mode imaging including CT, PA, and photothermal imaging, as well as high performance of photothermal therapeutic effect [102].