High-Throughput Synthesis of Nanogap-Rich Gold Nanoshells Using Dual-Channel Infusion System.

Gold nanoshells have been actively applied in industries beyond the research stage because of their unique optical properties. Although numerous methods have been reported for gold nanoshell synthesis, the labor-intensive and time-consuming production process is an issue that must be overcome to meet industrial demands. To resolve this, we report a high-throughput synthesis method for nanogap-rich gold nanoshells based on a core silica support (denoted as SiO2@Au NS), affording a 50-fold increase in scale by combining it with a dual-channel infusion pump system. By continuously dropping the reactant solution through the pump, nanoshells with closely packed Au nanoparticles were prepared without interparticle aggregation. The thickness of the gold nanoshells was precisely controlled at 2.3-17.2 nm by regulating the volume of the reactant solution added dropwise. Depending on the shell thickness, the plasmonic characteristics of SiO2@Au NS prepared by the proposed method could be tuned. Moreover, SiO2@Au NS exhibited surface-enhanced Raman scattering activity comparable to that of gold nanoshells prepared by a previously reported low-throughput method at the same reactant ratio. The results indicate that the proposed high-throughput synthesis method involving the use of a dual-channel infusion system will contribute to improving the productivity of SiO2@Au NS with tunable plasmonic characteristics.

Bifunctional Tumor-Targeted Bioprobe for Phothotheranosis.

Background: Near-infrared (NIR) phototheranostics provide promising noninvasive imaging and treatment for head and neck squamous cell carcinoma (HNSCC), capitalizing on its adjacency to skin or mucosal surfaces. Activated by laser irradiation, targeted NIR fluorophores can selectively eradicate cancer cells, harnessing the power of synergistic photodynamic therapy and photothermal therapy. However, there is a paucity of NIR bioprobes showing tumor-specific targeting and effective phototheranosis without hurting surrounding healthy tissues. Methods: We engineered a tumor-specific bifunctional NIR bioprobe designed to precisely target HNSCC and induce phototheranosis using bioconjugation of a cyclic arginine-glycine-aspartic acid (cRGD) motif and zwitterionic polymethine NIR fluorophore. The cytotoxic effects of cRGD-ZW800-PEG were measured by assessing heat and reactive oxygen species (ROS) generation upon an 808-nm laser irradiation. We then determined the in vivo efficacy of cRGD-ZW800-PEG in the FaDu xenograft mouse model of HNSCC, as well as its biodistribution and clearance, using a customized portable NIR imaging system. Results: Real-time NIR imaging revealed that intravenously administered cRGD-ZW800-PEG targeted tumors rapidly within 4 h postintravenous injection in tumor-bearing mice. Upon laser irradiation, cRGD-ZW800-PEG produced ROS and heat simultaneously and exhibited synergistic photothermal and photodynamic effects on the tumoral tissue without affecting the neighboring healthy tissues. Importantly, all unbound bioprobes were cleared through renal excretion. Conclusions: By harnessing phototheranosis in combination with tailored tumor selectivity, our targeted bioprobe ushers in a promising paradigm in cancer treatment. It promises safer and more efficacious therapeutic avenues against cancer, marking a substantial advancement in the field.

In vivo quantification of programmed death-ligand-1 expression heterogeneity in tumors using fluorescence lifetime imaging.

Cancer patient selection for immunotherapy is often based on programmed death-ligand-1 (PD-L1) expression as a biomarker. PD-L1 expression is currently quantified using immunohistochemistry, which can only provide snapshots of PD-L1 expression status in microscopic regions of ex vivo specimens. In vivo imaging using targeted agents can capture dynamic variations of PD-L1 expression in entire tumors within and across multiple subjects. Towards this goal, several PD-L1 targeted molecular imaging probes have been evaluated in murine models and humans. However, clinical translation of these probes has been limited due to a significant non-specific accumulation of the imaging probes and the inability of conventional imaging modalities to provide quantitative readouts that can be compared across multiple subjects. Here we report that in vivo time-domain (TD) fluorescence imaging can provide quantitative estimates of baseline tumor PD-L1 heterogeneity across untreated mice and variations in PD-L1 expression across mice undergoing clinically relevant anti-PD1 treatment. This approach relies on a significantly longer fluorescence lifetime (FLT) of PD-L1 specific anti-PD-L1 antibody tagged to IRDye 800CW (αPDL1-800) compared to nonspecific αPDL1-800. Leveraging this unique FLT contrast, we show that PD-L1 expression can be quantified across mice both in superficial breast tumors using planar FLT imaging, and in deep-seated liver tumors (>5 mm depth) using the asymptotic TD algorithm for fluorescence tomography. Our results suggest that FLT contrast can accelerate the preclinical investigation and clinical translation of novel molecular imaging probes by providing robust quantitative readouts of receptor expression that can be readily compared across subjects.

H-Dot Mediated Nanotherapeutics Mitigate Systemic Toxicity of Platinum-Based Anticancer Drugs.

Platinum-based anticancer agents have revolutionized oncological treatments globally. However, their therapeutic efficacy is often accompanied by systemic toxicity. Carboplatin, recognized for its relatively lower toxicity profile than cisplatin, still presents off-target toxicities, including dose-dependent cardiotoxicity, neurotoxicity, and myelosuppression. In this study, we demonstrate a delivery strategy of carboplatin to mitigate its off-target toxicity by leveraging the potential of zwitterionic nanocarrier, H-dot. The designed carboplatin/H-dot complex (Car/H-dot) exhibits rapid drug release kinetics and notable accumulation in proximity to tumor sites, indicative of amplified tumor targeting precision. Intriguingly, the Car/H-dot shows remarkable efficacy in eliminating tumors across insulinoma animal models. Encouragingly, concerns linked to carboplatin-induced cardiotoxicity are effectively alleviated by adopting the Car/H-dot nanotherapeutic approach. This pioneering investigation not only underscores the viability of H-dot as an organic nanocarrier for platinum drugs but also emphasizes its pivotal role in ameliorating associated toxicities. Thus, this study heralds a promising advancement in refining the therapeutic landscape of platinum-based chemotherapy.

Synthesis, Optical Properties, and In Vivo Biodistribution Performance of Polymethine Cyanine Fluorophores.

Near-infrared (NIR) cyanine dyes showed enhanced properties for biomedical imaging. A systematic modification within the cyanine skeleton has been made through a facile design and synthetic route for optimal bioimaging. Herein, we report the synthesis of 11 NIR cyanine fluorophores and an investigation of their physicochemical properties, optical characteristics, photostability, and in vivo performance. All synthesized fluorophores absorb and emit within 610-817 nm in various solvents. These dyes also showed high molar extinction coefficients ranging from 27,000 to 270,000 cm-1 M-1, quantum yields 0.01 to 0.33, and molecular brightness 208-79,664 cm-1 M-1 in the tested solvents. Photostability data demonstrate that all tested fluorophores 28, 18, 20, 19, 25, and 24 are more photostable than the FDA-approved indocyanine green. In the biodistribution study, most compounds showed tissue-specific targeting to selectively accumulate in the adrenal glands, lymph nodes, or gallbladder while excreted to the hepatobiliary clearance route. Among the tested, compound 23 showed the best targetability to the bone marrow and lymph nodes. Since the safety of cyanine fluorophores is well established, rationally designed cyanine fluorophores established in the current study will expand an inventory of contrast agents for NIR imaging of not only normal tissues but also cancerous regions originating from these organs/tissues.

Complement C1q essential for aeroallergen sensitization via CSF1R+ conventional dendritic cells type 2.

BACKGROUND: Dendritic cells (DCs) are heterogeneous, comprising multiple subsets with unique functional specifications. Our previous work has demonstrated that the specific conventional type 2 DC subset, CSF1R+cDC2s, plays a critical role in sensing aeroallergens. OBJECTIVE: It remains to be understood how CSF1R+cDC2s recognize inhaled allergens. We sought to elucidate the transcriptomic programs and receptor-ligand interactions essential for function of this subset in allergen sensitization. METHODS: We applied single-cell RNA sequencing to mouse lung DCs. Conventional DC-selective knockout mouse models were employed, and mice were subjected to inhaled allergen sensitization with multiple readouts of asthma pathology. Under the clinical arm of this work, human lung transcriptomic data were integrated with mouse data, and bronchoalveolar lavage (BAL) specimens were collected from subjects undergoing allergen provocation, with samples assayed for C1q. RESULTS: We found that C1q is selectively enriched in lung CSF1R+cDC2s, but not in other lung cDC2 or cDC1 subsets. Depletion of C1q in conventional DCs significantly attenuates allergen sensing and features of asthma. Additionally, we found that C1q binds directly to human dust mite allergen, and the C1q receptor CD91 (LRP1) is required for lung CSF1R+cDC2s to recognize the C1q-allergen complex and induce allergic lung inflammation. Lastly, C1q is enriched in human BAL samples following subsegmental allergen challenge, and human RNA sequencing data demonstrate close homology between lung IGSF21+DCs and mouse CSF1R+cDC2s. CONCLUSIONS: C1q is secreted from the CSF1R+cDC2 subset among conventional DCs. Our data indicate that the C1q-LRP1 axis represents a candidate for translational therapeutics in the prevention and suppression of allergic lung inflammation.

Silica Encapsulation of Hydrophobic Optical NP-Embedded Silica Particles with Trimethoxy(2-Phenylethyl)silane.

Nanoparticles (NP) with optical properties embedded silica particles have been widely used in various fields because of their unique properties. The surfaces of optical NPs have been modified with various organic ligands to maintain their unique optical properties and colloidal stability. Among the surface modification methods, silica encapsulation of optical NPs is widely used to enhance their biocompatibility and stability. However, in the case of NPs with hydrophobic ligands on the surface, the ligands that determine the optical properties of the NPs may detach from the NPs, thereby changing the optical properties during silica encapsulation. Herein, we report a generally applicable silica encapsulation method using trimethoxy(2-phenylethyl)silane (TMPS) for non-hydrophilic optical NPs, such as quantum dots (QDs) and gold NPs. This silica encapsulation method was applied to fabricate multiple silica-encapsulated QD-embedded silica NPs (SiO2@QD@SiO2 NPs; QD2) and multiple silica-encapsulated gold NP-embedded silica NPs labeled with 2-naphthalene thiol (SiO2@Au2-NT@SiO2). The fabricated silica-encapsulated NPs exhibited optical properties without significant changes in the quantum yield or Raman signal intensity.

Ultralow Background Near-Infrared Fluorophores with Dual-Channel Intraoperative Imaging Capability.

Two of the most pressing challenges facing bioimaging are nonspecific uptake of intravenously administered contrast agents and incomplete elimination of unbound targeted agents from the body. Designing a targeted contrast agent that shows fast clearance from background tissues and eventually the body after complete targeting is key to the success of image-guided interventions. Here, this work describes the development of renally clearable near-infrared contrast agents and their potential use for dual-channel image-guided tumor targeting. cRGD-ZW800-PEG (800 nm channel) and ZW700-PEG (700 nm channel) are able to visualize tumor margins and tumor vasculature simultaneously and respectively. These targeted agents show rapid elimination from the bloodstream, followed by renal clearance, which together significantly lower off-target background signals and potential toxicity. To demonstrate its applicability, this multispectral imaging is performed in various tumor-bearing animal models including lung cancer, pancreatic neuroendocrine tumors, breast, and ovarian cancer.

Optical Tissue Phantoms for Quantitative Evaluation of Surgical Imaging Devices.

Optical tissue phantoms (OTPs) have been extensively applied to the evaluation of imaging systems and surgical training. Due to their human tissue-mimicking characteristics, OTPs can provide accurate optical feedback on the performance of image-guided surgical instruments, simulating the biological sizes and shapes of human organs, and preserving similar haptic responses of original tissues. This review summarizes the essential components of OTPs (i.e., matrix, scattering and absorbing agents, and fluorophores) and the various manufacturing methods currently used to create suitable tissue-mimicking phantoms. As photobleaching is a major challenge in OTP fabrication and its feedback accuracy, phantom photostability and how the photobleaching phenomenon can affect their optical properties are discussed. Consequently, the need for novel photostable OTPs for the quantitative evaluation of surgical imaging devices is emphasized.

3D cell-printing of gradient multi-tissue interfaces for rotator cuff regeneration.

Owing to the prevalence of rotator cuff (RC) injuries and suboptimal healing outcome, rapid and functional regeneration of the tendon-bone interface (TBI) after RC repair continues to be a major clinical challenge. Given the essential role of the RC in shoulder movement, the engineering of biomimetic multi-tissue constructs presents an opportunity for complex TBI reconstruction after RC repair. Here, we propose a gradient cell-laden multi-tissue construct combined with compositional gradient TBI-specific bioinks via 3D cell-printing technology. In vitro studies demonstrated the capability of a gradient scaffold system in zone-specific inducibility and multi-tissue formation mimicking TBI. The regenerative performance of the gradient scaffold on RC regeneration was determined using a rat RC repair model. In particular, we adopted nondestructive, consecutive, and tissue-targeted near-infrared fluorescence imaging to visualize the direct anatomical change and the intricate RC regeneration progression in real time in vivo. Furthermore, the 3D cell-printed implant promotes effective restoration of shoulder locomotion function and accelerates TBI healing in vivo. In summary, this study identifies the therapeutic contribution of cell-printed constructs towards functional RC regeneration, demonstrating the translational potential of biomimetic gradient constructs for the clinical repair of multi-tissue interfaces.

HSA-ZW800-PEG for Enhanced Optophysical Stability and Tumor Targeting.

Small molecule fluorophores often face challenges such as short blood half-life, limited physicochemical and optical stability, and poor pharmacokinetics. To overcome these limitations, we conjugated the zwitterionic near-infrared fluorophore ZW800-PEG to human serum albumin (HSA), creating HSA-ZW800-PEG. This conjugation notably improves chemical, physical, and optical stability under physiological conditions, addressing issues commonly encountered with small molecules in biological applications. Additionally, the high molecular weight and extinction coefficient of HSA-ZW800-PEG enhances biodistribution and tumor targeting through the enhanced permeability and retention effect. The unique distribution and elimination dynamics, along with the significantly extended blood half-life of HSA-ZW800-PEG, contribute to improved tumor targetability in both subcutaneous and orthotopic xenograft tumor-bearing animal models. This modification not only influences the pharmacokinetic profile, affecting retention time and clearance patterns, but also enhances bioavailability for targeting tissues. Our study guides further development and optimization of targeted imaging agents and drug-delivery systems.

Pharmacokinetics and tissue distribution of deferoxamine-based nanochelator in rats.

Aim: To characterize the pharmacokinetics of deferoxamine-conjugated nanoparticles (DFO-NPs), a novel nanochelator for removing excess iron. Materials & methods: The pharmacokinetics of DFO-NPs were evaluated in Sprague-Dawley rats at three doses (3.3, 10 and 30 µmol/kg) after intravenous and subcutaneous administration. Results: DFO-NPs exhibited a biphasic concentration-time profile after intravenous administration with a short terminal half-life (2.0-3.2 h), dose-dependent clearance (0.111-0.179 l/h/kg), minimal tissue distribution and exclusive renal excretion with a possible saturable reabsorption mechanism. DFO-NPs after subcutaneous administration exhibited absorption-rate-limited kinetics with a prolonged half-life (5.7-10.1 h) and favorable bioavailability (47-107%). Conclusion: DFO-NPs exhibit nonlinear pharmacokinetics with increasing dose, and subcutaneous administration substantially improves drug exposure, thereby making it a clinically viable administration route for iron chelation.

Iron is an essential metal nutrient, but excess iron produces toxic effects that damage multiple organs including the heart, liver and pancreas. Deferoxamine (DFO) is a US FDA-approved drug for treating iron overload, but its use is limited by serious adverse effects and an inconvenient daily dose scheme. The recent development of a DFO-based nanomedicine (DFO-NP) has shown promise in treating iron overload in animals and was safer in animals. Before this new drug can be given to humans, how it is absorbed into the body, processed in the body and removed from the body when given in different amounts and dose routes must be determined. In this study, we tested the absorption, distribution and removal of DFO-NPs after intravenous and subcutaneous injection in rats. This study showed that DFO-NPs behave differently when changing the dose and that subcutaneous injection makes the drug stay in the body longer without ill effect, which means it could be given to patients this way.

Microwave-Assisted Synthesis of the Red-Shifted Pentamethine Tetrahydroxanthylium Core with Absorbance within the Near Infrared-II Window.

Thirteen red-shifted pentamethine dimethyl and diethylamino tetrahydroxanthylium derivatives have been successfully synthesized via the microwave-assisted approach. The optimized conditions developed in the synthesis provided an excellent yield in expedited reaction time. These newly synthesized dyes show well-defined optical properties resulting from the diverse substitutions at the central meso positions. The majority of the compounds have a maximum wavelength of absorbance within 946-1022 nm with extinction coefficients in the range of 9700-110,680 M-1 cm-1 in various solvents such as MeOH, EtOH, DMSO, DCM, MeCN, and DMF. These fluorophores, to the best of our knowledge, are the first NIR-II small molecules synthesized using microwave chemistry. We also investigated these dyes for their NIR fluorescence imaging capabilities. Diethylamino-substituted compounds and bromination resulted in higher uptake in the adrenal gland compared to dimethylamino fluorophores. In addition, micellar structures of compounds 7 and 15 improved the targetability of the original dyes to the bone marrow, lymph nodes, and nerves. Overall, NIR-II imaging has the potential to visualize biologically targeted tissues in living organisms.

P800SO3-PEG: a renal clearable bone-targeted fluorophore for theranostic imaging.

BACKGROUND: Due to the deep tissue penetration and reduced scattering, NIR-II fluorescence imaging is advantageous over conventional visible and NIR-I fluorescence imaging for the detection of bone growth, metabolism, metastasis, and other bone-related diseases. METHODS: Bone-targeted heptamethine cyanine fluorophores were synthesized by substituting the meso-carbon with a sulfur atom, resulting in a bathochromic shift and increased fluorescence intensity. The physicochemical, optical, and thermal stability of newly synthesized bone-targeted NIR fluorophores was performed in aqueous solvents. Calcium binding, bone-specific targeting, biodistribution, pharmacokinetics, and 2D and 3D NIR imaging were performed in animal models. RESULTS: The newly synthesized S-substituted heptamethine fluorophores demonstrated a high affinity for hydroxyapatite and calcium phosphate, which improved bone-specific targeting with signal-background ratios > 3.5. Particularly, P800SO3-PEG showed minimum nonspecific uptake, and most unbound molecules were excreted into the urinary bladder. Histological analyses demonstrated that P800SO3-PEG remained stable in the bone for over two weeks and was incorporated into bone matrices. Interestingly, the flexible thiol ethylene glycol linker on P800SO3-PEG induced a promising photothermal effect upon NIR laser irradiation, demonstrating potential theranostic imaging. CONCLUSIONS: P800SO3-PEG shows a high affinity for bone tissues, deeper tissue imaging capabilities, minimum nonspecific uptake in the major organs, and photothermal effect upon laser irradiation, making it optimal for bone-targeted theranostic imaging.

Silica Shell Thickness-Dependent Fluorescence Properties of SiO2@Ag@SiO2@QDs Nanocomposites.

Silica shell coatings, which constitute important technology for nanoparticle (NP) developments, are utilized in many applications. The silica shell's thickness greatly affects distance-dependent optical properties, such as metal-enhanced fluorescence (MEF) and fluorescence quenching in plasmonic nanocomposites. However, the precise control of silica-shell thicknesses has been mainly conducted on single metal NPs, and rarely on complex nanocomposites. In this study, silica shell-coated Ag nanoparticle-assembled silica nanoparticles (SiO2@Ag@SiO2), with finely controlled silica shell thicknesses (4 nm to 38 nm), were prepared, and quantum dots (QDs) were introduced onto SiO2@Ag@SiO2. The dominant effect between plasmonic quenching and MEF was defined depending on the thickness of the silica shell between Ag and QDs. When the distance between Ag NPs to QDs was less than ~10 nm, SiO2@Ag@SiO2@QDs showed weaker fluorescence intensities than SiO2@QD (without metal) due to the quenching effect. On the other hand, when the distance between Ag NPs to QDs was from 10 nm to 14 nm, the fluorescence intensity of SiO2@Ag@SiO2@QD was stronger than SiO2@QDs due to MEF. The results provide background knowledge for controlling the thickness of silica shells in metal-containing nanocomposites and facilitate the development of potential applications utilizing the optimal plasmonic phenomenon.

Phototheranostics for multifunctional treatment of cancer with fluorescence imaging.

Phototheranostics stem from the recent advances in nanomedicines and bioimaging to diagnose and treat human diseases. Since tumors' diversity, heterogeneity, and instability limit the clinical application of traditional diagnostics and therapeutics, phototheranostics, which combine light-induced therapeutic and diagnostic modalities in a single platform, have been widely investigated. Numerous efforts have been made to develop phototheranostics for efficient light-induced antitumor therapeutics with minimal side effects. Herein, we review the fundamentals of phototheranostic nanomedicines with their biomedical applications. Furthermore, the progress of near-infrared fluorescence imaging and cancer treatments, including photodynamic therapy and photothermal therapy, along with chemotherapy, immunotherapy, and gene therapy, are summarized. This review also discusses the opportunities and challenges associated with the clinical translation of phototheranostics in pan-cancer research. Phototheranostics can pave the way for future research, improve the quality of life, and prolong cancer patients' survival times.

NIR fluorescence imaging and treatment for cancer immunotherapy.

Cancer immunotherapy has emerged as one of the most powerful anticancer therapies. However, the details on the interaction between tumors and the immune system are complicated and still poorly understood. Optical fluorescence imaging is a technique that allows for the visualization of fluorescence-labeled immune cells and monitoring of the immune response during immunotherapy. To this end, near-infrared (NIR) light has been adapted for optical fluorescence imaging because it is relatively safe and simple without hazardous ionizing radiation and has relatively deeper tissue penetration into living organisms than visible fluorescence light. In this review, we discuss state-of-the-art NIR optical imaging techniques in cancer immunotherapy to observe the dynamics, efficacy, and responses of the immune components in living organisms. The use of bioimaging labeling techniques will give us an understanding of how the immune system is primed and ultimately developed.

Image-guided drug delivery of nanotheranostics for targeted lung cancer therapy.

Enormous efforts have been made to integrate various therapeutic interventions into multifunctional nanoplatforms, resulting in the advance of nanomedicine. Image-guided drug delivery plays a pivotal role in this field by providing specific targeting as well as image navigation for disease prognosis. Methods: We demonstrate image-guided surgery and drug delivery for the treatment of lung cancer using nanotheranostic H-dots loaded with gefitinib and genistein. Results: The surgical margin for lung tumors is determined by image guidance for precise tumor resection, while targeted anti-cancer drugs function simultaneously for synergistic combination therapy. Compared to conventional chemotherapies, H-dot complexes could improve the therapeutic efficacy of drugs while reducing the risk of adverse effects and drug resistance owing to their ideal biodistribution profiles, high targetability, low nonspecific tissue uptake, and fast renal excretion. Conclusions: These H-dot complexes have unlocked a unique framework for integrating multiple therapeutic and diagnostic modalities into one nanoplatform.

Topical pH Sensing NIR Fluorophores for Intraoperative Imaging and Surgery of Disseminated Ovarian Cancer.

Fluorescence-guided surgery (FGS) aids surgeons with real-time visualization of small cancer foci and borders, which improves surgical and prognostic efficacy of cancer. Despite the steady advances in imaging devices, there is a scarcity of fluorophores available to achieve optimal FGS. Here, 1) a pH-sensitive near-infrared fluorophore that exhibits rapid signal changes in acidic tumor microenvironments (TME) caused by the attenuation of intramolecular quenching, 2) the inherent targeting for cancer based on chemical structure (structure inherent targeting, SIT), and 3) mitochondrial and lysosomal retention are reported. After topical application of PH08 on peritoneal tumor regions in ovarian cancer-bearing mice, a rapid fluorescence increase (< 10 min), and extended preservation of signals (> 4 h post-topical application) are observed, which together allow for the visualization of submillimeter tumors with a high tumor-to-background ratio (TBR > 5.0). In addition, PH08 is preferentially transported to cancer cells via organic anion transporter peptides (OATPs) and colocalizes in the mitochondria and lysosomes due to the positive charges, enabling a long retention time during FGS. PH08 not only has a significant impact on surgical and diagnostic applications but also provides an effective and scalable strategy to design therapeutic agents for a wide array of cancers.

Highly sensitive near-infrared SERS nanoprobes for in vivo imaging using gold-assembled silica nanoparticles with controllable nanogaps.

BACKGROUND: To take advantages, such as multiplex capacity, non-photobleaching property, and high sensitivity, of surface-enhanced Raman scattering (SERS)-based in vivo imaging, development of highly enhanced SERS nanoprobes in near-infrared (NIR) region is needed. A well-controlled morphology and biocompatibility are essential features of NIR SERS nanoprobes. Gold (Au)-assembled nanostructures with controllable nanogaps with highly enhanced SERS signals within multiple hotspots could be a breakthrough. RESULTS: Au-assembled silica (SiO2) nanoparticles (NPs) (SiO2@Au@Au NPs) as NIR SERS nanoprobes are synthesized using the seed-mediated growth method. SiO2@Au@Au NPs using six different sizes of Au NPs (SiO2@Au@Au50-SiO2@Au@Au500) were prepared by controlling the concentration of Au precursor in the growth step. The nanogaps between Au NPs on the SiO2 surface could be controlled from 4.16 to 0.98 nm by adjusting the concentration of Au precursor (hence increasing Au NP sizes), which resulted in the formation of effective SERS hotspots. SiO2@Au@Au500 NPs with a 0.98-nm gap showed a high SERS enhancement factor of approximately 3.8 × 106 under 785-nm photoexcitation. SiO2@Au@Au500 nanoprobes showed detectable in vivo SERS signals at a concentration of 16 µg/mL in animal tissue specimen at a depth of 7 mm. SiO2@Au@Au500 NPs with 14 different Raman label compounds exhibited distinct SERS signals upon subcutaneous injection into nude mice. CONCLUSIONS: SiO2@Au@Au NPs showed high potential for in vivo applications as multiplex nanoprobes with high SERS sensitivity in the NIR region.