Lighting Up MicroRNA in Living Cells by the Disassembly of Lock‐Like DNA‐Programmed UCNPs‐AuNPs through the Target Cycling Amplification Strategy

Intracellular microRNAs imaging based on upconversion nanoprobes has great potential in cancer diagnostics and treatments. However, the relatively low detection sensitivity limits their application. Herein, a lock‐like DNA (LLD) generated by a hairpin DNA (H1) hybridizing with a bolt DNA (bDNA) sequence is designed, which is used to program upconversion nanoparticles (UCNPs, NaYF₄@NaYF₄:Yb, Er@NaYF₄) and gold nanoparticles (AuNPs). The upconversion emission is quenched through luminescence resonance energy transfer (LRET). The multiple LLD can be repeatedly opened by one copy of target microRNA under the aid of fuel hairpin DNA strands (H2) to trigger disassembly of AuNPs from the UCNP, resulting in the lighting up of UCNPs with a high detection signal gain. This strategy is verified using microRNA‐21 as model. The expression level of microRNA‐21 in various cells lines can be sensitively measured in vitro, meanwhile cancer cells and normal cells can be easily and accurately distinguished by intracellular microRNA‐21 imaging via the nanoprobes. The detection limit is about 1000 times lower than that of the previously reported upconversion nanoprobes without signal amplification. This is the first time a nonenzymatic signal amplification method has been combined with UCNPs for imaging intracellular microRNAs, which has great potential for cancer diagnosis.     

Zinc‐Dithizone Complex Engineered Upconverting Nanosensors for the Detection of Hypochlorite in Living Cells

Current chemo/biosensors for hypochlorous acid or hypochlorite detections are usually limited to the submicromolar level because of their insufficient sensitivity, which is a problem because the concentrations in biological matrices is generally on the nanomolar scale or even lower. Developing a probe with a high enough sensitivity remains a challenge. Using the minimal background fluorescence of upconversion nanocrystals to our advantage, we herein report on an energy‐transfer mechanism‐based upconversion luminescent nanosensor for the sensitive and selective detection of hypochlorite in aqueous solution. In this nanosensor water‐dispersible upconversion nanoparticles act as the energy donor and a novel hypochlorite‐responsive coordination complex Zn(DZ)₃ is employed as the energy acceptor. The quenched upconversion luminescence, induced by the Zn(DZ)₃ complex, can be efficiently recovered after addition of hypochlorite through the selective oxidative breakage of the Zn‐S‐C bonds in the Zn(DZ)₃ complex, which was verified by mass spectrometry. The detection limit for hypochlorite of this sensing system is as low as 3 nM. Furthermore, this newly coordination‐complex engineered upconversion nanosensor is successfully applied to image different amounts of exogenous hypochlorite in living HeLa cells.     

Disposable 3D GNAs/AuNPs DNA‐Circuit Strip for miRNAs Dynamic Quantification

Real‐time quantitative monitoring of miRNAs plays an essential role in diagnosis and therapeutics. Herein, a DSN‐coupled graphene nanoarray/gold nanoparticles (GNAs/AuNPs) carbon paper (CP) electrode for the dynamic, sensitive, and real‐time analysis of miRNAs is reported. GNAs are vertically grown on the conductive CP by radio frequency plasma enhanced chemical vapor deposition, and AuNPs are electrodeposited on CP/GNAs to build a 3D ultrasensitive sensing interface with large specific surface area, good conductivity and biocompatibility. The dynamic quantitative monitoring of microRNA‐21 (miR‐21) is realized by cyclic voltammetry with a series of different concentrations within 16 min, and this 3D GNAs/AuNPs DNA‐circuit strip shows good performance for the simultaneous detection of miR‐21 and miR‐155, and the detection limits are as low as 21.4 and 30.3 am, respectively. Moreover, comparable detection results are achieved for clinical samples between the proposed sensor and qRT‐PCR, suggesting the reliability of the constructed sensor. This ultrasensitive sensing and disposable DNA‐circuit strip with 3D structure can efficiently shorten the diffusion distance between reactive biomolecules and the sensing interface, enhance the hybridization of probes and improve the sensitivity of the biosensor, holding great promise for the rapid, quantitative and dynamic monitoring of multiple low concentrations of biomolecules in point‐of‐care clinical analysis.     

pH‐Responsive PEG‐Shedding and Targeting Peptide‐Modified Nanoparticles for Dual‐Delivery of Irinotecan and microRNA to Enhance Tumor‐Specific Therapy

Irinotecan is one of the main chemotherapeutic agents for colorectal cancer (CRC). MicroRNA‐200 (miR‐200) has been reported to inhibit metastasis in cancer cells. Herein, pH‐sensitive and peptide‐modified liposomes and solid lipid nanoparticles (SLN) are designed for encapsulation of irinotecan and miR‐200, respectively. These peptides include one cell‐penetrating peptide, one ligand targeted to tumor neovasculature undergoing angiogenesis, and one mitochondria‐targeting peptide. The peptide‐modified nanoparticles are further coated with a pH‐sensitive PEG‐lipid derivative with an imine bond. These specially‐designed nanoparticles exhibit pH‐responsive release, internalization, and intracellular distribution in acidic pH of colon cancer HCT116 cells. These nanoparticles display low toxicity to blood and noncancerous intestinal cells. Delivery of miR‐200 by SLN further increases the cytotoxicity of irinotecan‐loaded liposomes against CRC cells by triggering apoptosis and suppressing RAS/β‐catenin/ZEB/multiple drug resistance (MDR) pathways. Using CRC‐bearing mice, the in vivo results further indicate that irinotecan and miR‐200 in pH‐responsive targeting nanoparticles exhibit positive therapeutic outcomes by inhibiting colorectal tumor growth and reducing systemic toxicity. Overall, successful delivery of miR and chemotherapy by multifunctional nanoparticles may modulate β‐catenin/MDR/apoptosis/metastasis signaling pathways and induce programmed cancer cell death. Thus, these pH‐responsive targeting nanoparticles may provide a potential regimen for effective treatment of colorectal cancer.     

Albumin Nanoshell Encapsulation of Near‐Infrared‐Excitable Rare‐Earth Nanoparticles Enhances Biocompatibility and Enables Targeted Cell Imaging

The use of traditional fluorophores for in vivo imaging applications is limited by poor quantum yield, poor tissue penetration of the excitation light, and excessive tissue autofluorescence, while the use of inorganic fluorescent particles that offer a high quantum yield is frequently limited due to particle toxicity. Rare‐earth‐doped nanoparticles that utilize near‐infrared upconversion overcome the optical limitations of traditional fluorophores, but are not typically suitable for biological application due to their insolubility in aqueous solution, lack of functional surface groups for conjugation of biomolecules, and potential cytotoxicity. A new approach to establish highly biocompatible and biologically targetable nanoshell complexes of luminescent rare‐earth‐doped NaYF₄ nanoparticles (REs) excitable with 920–980 nm near‐infrared light for biomedical imaging applications is reported. The approach involves the encapsulation of NaYF₄ nanoparticles doped with Yb and Er within human serum albumin nanoshells to create water‐dispersible, biologically functionalizable composite particles. These particles exhibit narrow size distributions around 200 nm and are stable in aqueous solution for over 4 weeks. The albumin shell confers cytoprotection and significantly enhances the biocompatibility of REs even at concentrations above 200 µg REs mL^?1. Composite particles conjugated with cyclic arginine‐glycine‐aspartic acid (cRGD) specifically target both human glioblastoma cell lines and melanoma cells expressing α_vβ₃ integrin receptors. These findings highlight the promise of albumin‐encapsulated rare‐earth nanoparticles for imaging cancer cells in vitro and the potential for targeted imaging of disease sites in vivo.     

Iridium(III) Complex–Derived Polymeric Micelles with Low Dark Toxicity and Strong NIR Excitation for Phototherapy and Chemotherapy

Iridium(III) complexes are potent candidates for photodynamic therapy. However, their clinical usage is impeded by their poor water solubility, high dark toxicity, and negligible absorption in near‐infrared region (NIR region). Here, it is proposed to solve these challenges by developing an iridium(III) complexe‐based polymeric micelle system. This system is self‐assembled using an iridium(III) complex‐containing amphiphilic block polymer. The upconversion nanoparticles are included in the polymeric micelles to permit NIR excitation. Compared with the nonformulated iridium(III) complexes, under NIR stimulation, this polymeric micelle system exhibits higher ¹O₂ generation efficiency, negligible dark toxicity, excellent tumor‐targeting ability, and synergistic phototherapy–chemotherapy effect both in vitro and in vivo.     

A Modular System for the Design of Stimuli‐Responsive Multifunctional Nanoparticle Aggregates by Use of Host–Guest Chemistry

A self‐assembly approach for the design of multifunctional nanomaterials consisting of different nanoparticles (gold, iron oxide, and lanthanide‐doped LiYF₄) is developed. This modular system takes advantage of the light‐responsive supramolecular host–guest chemistry of β‐cyclodextrin and arylazopyrazole, which enables the dynamic and reversible self‐assembly of particles to spherical nanoparticle aggregates in aqueous solution. Due to the magnetic iron oxide nanoparticles, the aggregates can be manipulated by an external magnetic field leading to the formation of linear structures. As a result of the integration of upconversion nanoparticles, the aggregates are additionally responsive to near‐infrared light and can be redispersed by use of the upconversion effect. By varying the nanoparticle and linker concentrations the composition, size, shape, and properties of the multifunctional nanoparticle aggregates can be fine‐tuned.     

miRNA‐Specific Unlocking of Drug‐Loaded Metal–Organic Framework Nanoparticles: Targeted Cytotoxicity toward Cancer Cells

UiO‐68 metal–organic framework nanoparticles (NMOFs) are loaded with a doxorubicin drug (fluorescent dye analogs) and locked by means of structurally engineered duplex nucleic acid structures, where one strand is covalently linked to the NMOFs and the second strand is hybridized with the anchor strand. Besides the complementarity of the second strand to the anchor sequence, it includes the complementary sequence to the microRNAs (miRNA)‐21 or miRNA‐221 that is specific miRNA biomarker for MCF‐7 breast cancer cells or OVCAR‐3 ovarian cancer cells. In the presence of the respective miRNA biomarkers, the miRNA‐induced displacement of the strand associated with the anchor strand proceeds, resulting in the release of DNA/miRNA duplexes. The released duplexes are, however, engineered to be digested in the presence of exonuclease III, Exo III, a process that recycles the miRNAs and provides the autonomous amplified unlocking of the NMOFs and the release of the doxorubicin load (or the fluorescent dye analogs) even at low concentrations of miRNA. Preliminary cell experiments reveal that the respective NMOFs are unlocked by the miRNA‐21 or miRNA‐221, resulting in selective cytotoxicity toward MCF‐7 breast cancer cells or OVCAR‐3 ovarian cancer cells.     

Synthesis and Assembly of Click‐Nucleic‐Acid‐Containing PEG–PLGA Nanoparticles for DNA Delivery

Co‐delivery of both chemotherapy drugs and siRNA from a single delivery vehicle can have a significant impact on cancer therapy due to the potential for overcoming issues such as drug resistance. However, the inherent chemical differences between charged nucleic acids and hydrophobic drugs have hindered entrapment of both components within a single carrier. While poly(ethylene glycol)‐block‐poly(lactic‐co‐glycolic acid) (PEG–PLGA) copolymers have been used successfully for targeted delivery of chemotherapy drugs, loading of DNA or RNA has been poor. It is demonstrated that significant amounts of DNA can be encapsulated within PLGA‐containing nanoparticles through the use of a new synthetic DNA analog, click nucleic acids (CNAs). First, triblock copolymers of PEG‐CNA‐PLGA are synthesized and then formulated into polymer nanoparticles from oil‐in‐water emulsions. The CNA‐containing particles show high encapsulation of DNA complementary to the CNA sequence, whereas PEG‐PLGA alone shows minimal DNA loading, and non‐complementary DNA strands do not get encapsulated within the PEG‐CNA‐PLGA nanoparticles. Furthermore, the dye pyrene can be successfully co‐loaded with DNA and lastly, a complex, larger DNA sequence that contains an overhang complementary to the CNA can also be encapsulated, demonstrating the potential utility of the CNA‐containing particles as carriers for chemotherapy agents and gene silencers.     

A Chiral‐Nanoassemblies‐Enabled Strategy for Simultaneously Profiling Surface Glycoprotein and MicroRNA in Living Cells

Assemblies of nanomaterials for biological applications in living cells have attracted much attention. Herein, graphene oxide (GO)–gold nanoparticle (Au NP) assemblies are driven by a splint DNA strand, which is designed with two regions at both ends that are complementary with the DNA sequence anchored on the surface of the GO and the Au NPs. In the presence of microRNA (miR)‐21 and epithelial cell‐adhesion molecule (EpCAM), the hybridization of miR‐21 with a molecular probe leads to the separation of 6‐fluorescein‐phosphoramidite‐modified Au NPs from GO, resulting in a decrease in the Raman signal, while EpCAM recognition reduces circular dichroism (CD) signals. The CD signals reverse from negative in original assemblies into positive when reacted with cells, which correlates with two enantiomer geometries. The EpCAM detection has a good linear range of 8.47–74.78 pg mL^?1 and a limit of detection (LOD) of 3.63 pg mL^?1, whereas miR‐21 detection displays an outstanding linear range of 0.07–13.68 amol ng^?1_RNA and LOD of 0.03 amol ng^?1_RNA. All the results are in good agreement with those of the Raman and confocal bioimaging. The strategy opens up an avenue to allow the highly accurate and reliable diagnosis (dual targets) of clinic diseases.     

Pd nanowires as new biosensing materials for magnified fluorescent detection of nucleic acid

The designed synthesis of new nanomaterials with controlled shape, composition, and structure is critical for tuning their physical and chemical properties, and further developing interesting analytical sensing devices. Herein, we presented that Pd nanowires (NWs) can be used as a new biosensing platform for high-sensitivity nucleic acid detection. The general sensing concept is based on the fact that Pd NWs can adsorb the fluorescently labeled single-stranded DNA probe and lead to substantial fluorescence quenching of dye, followed by specific hybridization with the complementary region of the target DNA sequence. This results in desorption of double-stranded DNA from Pd NWs surface and subsequent recovery of fluorescence. Furthermore, an amplification strategy based on Pd NWs for nucleic acid detection by using exonuclease III (Exo III) was demonstrated. The present dual-magnification sensing system combined Pd NWs with Exo III has a detection range of 1.0 nM to 2.0 μM with the detection limit of 0.3 nM (S/N = 3), which is about 20-fold higher than that of traditional unamplified homogeneous assays.     

Supramolecular Recognition‐Mediated Layer‐by‐Layer Self‐Assembled Gold Nanoparticles for Customized Sensitivity in Paper‐Based Strip Nanobiosensors

Herein, a smart supramolecular self‐assembly‐mediated signal amplification strategy is developed on a paper‐based nanobiosensor to achieve the sensitive and customized detection of biomarkers. The host–guest recognition between β‐cyclodextrin‐coated gold nanoparticles (AuNPs) and 1‐adamantane acetic acid or tetrakis(4‐carboxyphenyl)porphyrin is designed and applied to the layer‐by‐layer self‐assembly of AuNPs at the test area of the strip. Thus, the amplified platform exhibits a high sensitivity with a detection limit at subattogram levels (approximately dozens of molecules per strip) and a wide dynamic range of concentration over seven orders of magnitude. The applicability and universality of this sensitive platform are demonstrated in clinically significant ranges to measure carcinoembryonic antigen and HIV‐1 capsid p24 antigen in spiked serum and clinical samples. The customized biomarker detection ability for the on‐demand needs of clinicians is further verified through cycle incubation‐mediated controllable self‐assembly. Collectively, the supramolecular self‐assembly amplification method is suitable as a universal point‐of‐care diagnostic tool and can be readily adapted as a platform technology for the sensitive assay of many different target analytes.     

Labeling TiO2 Nanoparticles with Dyes for Optical Fluorescence Microscopy and Determination of TiO2–DNA Nanoconjugate Stability

Visualization of nanoparticles without intrinsic optical fluorescence properties is a significant problem when performing intracellular studies. Such is the case with titanium dioxide (TiO₂) nanoparticles. These nanoparticles, when electronically linked to single‐stranded DNA oligonucleotides, have been proposed to be used both as gene knockout devices and as possible tumor imaging agents. By interacting with complementary target sequences in living cells, these photoinducible TiO₂–DNA nanoconjugates have the potential to cleave intracellular genomic DNA in a sequence specific and inducible manner. The nanoconjugates also become detectable by magnetic resonance imaging with the addition of gadolinium Gd(III) contrast agents. Herein two approaches for labeling TiO₂ nanoparticles and TiO₂–DNA nanoconjugates with optically fluorescent agents are described. This permits direct quantification of fluorescently labeled TiO₂ nanoparticle uptake in a large population of living cells (>10⁴ cells). X‐ray fluorescence microscopy (XFM) is combined with fluorescent microscopy to determine the relative intracellular stability of the nanoconjugates and used to quantify intracellular nanoparticles. Imaging the DNA component of the TiO₂–DNA nanoconjugate by fluorescent confocal microscopy within the same cell shows an overlap with the titanium signal as mapped by XFM. This strongly implies the intracellular integrity of the TiO₂–DNA nanoconjugates in malignant cells.     

Emerging ≈800 nm Excited Lanthanide‐Doped Upconversion Nanoparticles

Lanthanide‐doped upconversion nanoparticles can tune near‐infrared light to visible or even ultra‐violet light in emissions. Due to their unique photophysical and photochemical properties, as well as their promising bioapplications, there has been a great deal of enthusiastic research performed to study the properties of lanthanide‐doped upconversion nanoparticles in the past few years. Despite the considerable progress in this area, numerous challenges associated with the nanoparticles, such as a low upconversion efficiency, limited host materials, and a confined excitation wavelength, still remain, thus hindering further development with respect to their applications and in fundamental science. Recently, innovative strategies that utilize alternative sensitizers have been designed in order to engineer the excitation wavelengths of upconversion nanoparticles. Here, focusing on the excitation wavelength at ≈800 nm, recent advances in the design, property tuning, and applications of ≈800 nm excited upconversion nanoparticles are summarized. Benefiting from the unique features of ≈800 nm light, including deep tissue penetration depth and low photothermal effect, the ≈800 nm excited upconversion nanoparticles exhibit superior potential for biosensing, bioimaging, drug delivery, therapy, and three dimensional displays. The critical aspects of such emerging nanoparticles with regards to meeting the ever‐changing needs of future development are also discussed.     

Persistency of Enlarged Autolysosomes Underscores Nanoparticle‐Induced Autophagy in Hepatocytes

The diverse biological effects of nanomaterials form the basis for their applications in biomedicine but also cause safety issues. Induction of autophagy is a cellular response after nanoparticles exposure. It may be beneficial in some circumstances, yet autophagy‐mediated toxicity raises an alarming concern. Previously, it has been reported that upconversion nanoparticles (UCNs) elicit liver damage, with autophagy contributing most of this toxicity. However, the detailed mechanism is unclear. This study reveals persistent presence of enlarged autolysosomes in hepatocytes after exposure to UCNs and SiO₂ nanoparticles both in vitro and in vivo. This phenomenon is due to anomaly in the autophagy termination process named autophagic lysosome reformation (ALR). Phosphatidylinositol 4‐phosphate (PI(4)P) relocates onto autolysosome membrane, which is a key event of ALR. PI(4)P is then converted into phosphatidylinositol 4,5‐bisphosphate (PI(4,5)P₂) by phosphatidylinositol‐4‐phosphate 5‐kinase. Clathrin is subsequently recruited by PI(4,5)P₂ and leads to tubule budding of ALR. Yet it is observed that PI(4)P cannot be converted in nanoparticle‐treated hepatocytes cells. Exogenous supplement of PI(4,5)P₂ suppresses the enlarged autolysosomes in vitro. Abolishment of these enlarged autolysosomes by autophagy inhibitor relieves the hepatotoxicity of UCNs in vivo. The results provide evidence for disrupted ALR in nanoparticle‐treated hepatocytes, suggesting that the termination of nanoparticle‐induced autophagy is of equal importance as the initiation.     

Glutathione Mediated Size‐Tunable UCNPs‐Pt(IV)‐ZnFe2O4 Nanocomposite for Multiple Bioimaging Guided Synergetic Therapy

Here a multifunctional nanoplatform (upconversion nanoparticles (UCNPs)‐platinum(IV) (Pt(IV))?ZnFe₂O₄, denoted as UCPZ) is designed for collaborative cancer treatment, including photodynamic therapy (PDT), chemotherapy, and Fenton reaction. In the system, the UCNPs triggered by near‐infrared light can convert low energy photons to high energy ones, which act as the UV–vis source to simultaneously mediate the PDT effect and Fenton's reaction of ZnFe₂O₄ nanoparticles. Meanwhile, the Pt(IV) prodrugs can be reduced to high virulent Pt(II) by glutathione in the cancer cells, which can bond to DNA and inhibit the copy of DNA. The synergistic therapeutic effect is verified in vitro and in vivo results. The cleavage of Pt(IV) from UCNPs during the reduction process can shift the larger UCPZ nanoparticles (NPs) to the smaller ones, which promotes the enhanced permeability and retention (EPR) and deep tumor penetration. In addition, due to the inherent upconversion luminescence (UCL) and the doped Yb³⁺ and Fe³⁺ in UCPZ, this system can serve as a multimodality bioimaging contrast agent, covering UCL, X‐ray computed tomography, magnetic resonance imaging, and photoacoustic. A smart all‐in‐one imaging‐guided diagnosis and treatment system is realized, which should have a potential value in the treatment of tumor.     

Combined Replenishment of miR‐34a and let‐7b by Targeted Nanoparticles Inhibits Tumor Growth in Neuroblastoma Preclinical Models

Neuroblastoma (NB) tumor substantially contributes to childhood cancer mortality. The design of novel drugs targeted to specific molecular alterations becomes mandatory, especially for high‐risk patients burdened by chemoresistant relapse. The dysregulated expression of MYCN, ALK, and LIN28B and the diminished levels of miR‐34a and let‐7b are oncogenic in NB. Due to the ability of miRNA‐mimics to recover the tumor suppression functions of miRNAs underexpressed into cancer cells, safe and efficient nanocarriers selectively targeted to NB cells and tested in clinically relevant mouse models are developed. The technology exploits the nucleic acids negative charges to build coated‐cationic liposomes, then functionalized with antibodies against GD₂ receptor. The replenishment of miR‐34a and let‐7b by NB‐targeted nanoparticles, individually and more powerfully in combination, significantly reduces cell division, proliferation, neoangiogenesis, tumor growth and burden, and induces apoptosis in orthotopic xenografts and improves mice survival in pseudometastatic models. These functional effects highlight a cooperative down‐modulation of MYCN and its down‐stream targets, ALK and LIN28B, exerted by miR‐34a and let‐7b that reactivate regulatory networks leading to a favorable therapeutic response. These findings demonstrate a promising therapeutic efficacy of miR‐34a and let‐7b combined replacement and support its clinical application as adjuvant therapy for high‐risk NB patients.     

Gold and Hairpin DNA Functionalization of Upconversion Nanocrystals for Imaging and In Vivo Drug Delivery

Although multifunctional upconversion imaging probes have recently attracted considerable interest in biomedical research, there are currently few methods for stabilizing these luminescent nanoprobes with oligonucleotides in biological systems. Herein, a method to robustly disperse upconversion nanoprobes in physiological buffers based on rational design and synthesis of nanoconjugates comprising hairpin‐DNA‐modified gold nanoparticles is presented. This approach imparts the upconversion nanoprobes with excellent biocompatibility and circumvents the problem of particle agglomeration. By combining single‐band anti‐Stokes near‐infrared emission and the photothermal effect mediated by the coupling of gold to upconversion nanoparticles, a simple, versatile nanoparticulate system for simultaneous deep‐tissue imaging and drug molecule release in vivo is demonstrated.     

Simple and Rapid High‐Yield Synthesis and Size Sorting of Multibranched Hollow Gold Nanoparticles with Highly Tunable NIR Plasmon Resonances

Branched gold nanoparticles with sharp tips are considered excellent candidates for sensing and field enhancement applications. Here, a rapid and simple synthesis strategy is presented that generates highly branched gold nanoparticles with hollow cores and a ca.100% yield through a simple one‐pot seedless reaction at room temperature in the presence of Triton X‐100. It is shown that multibranched hollow gold nanoparticles of tunable dimensions, branch density and branch length can be obtained by adjusting the concentrations of the reactants. Insights into the formation mechanism point toward an aggregative type of growth involving hollow core formation first, and branching thereafter. The pronounced near‐infrared (NIR) plasmon band of the nanoparticles is due to the combined contribution from hollowness and branching, and can be tuned over a wide range (≈700–2000 nm). It is also demonstrated that the high environmental sensitivity of colloidal dispersions based on multibranched hollow gold nanoparticles can be boosted even further by separating the nanoparticles into fractions of given sizes and improved monodispersity by means of a glycerol density gradient. The possibility to obtain highly monodisperse multibranched hollow gold nanoparticles with predictable dimensions (50–300 nm) and branching and, therefore, tailored NIR plasmonic properties, highlights their potential for theranostic applications.