A black phosphorus nanosheet-based RNA delivery system for prostate cancer therapy by increasing the expression level of tumor suppressor gene PTEN via CeRNA mechanism.

BACKGROUND: Prostate cancer (PCa) has a high incidence in men worldwide, and almost all PCa patients progress to the androgen-independent stage which lacks effective treatment measures. PTENP1, a long non-coding RNA, has been shown to suppress tumor growth through the rescuing of PTEN expression via a competitive endogenous RNA (ceRNA) mechanism. However, PTENP1 was limited to be applied in the treatment of PCa for the reason of rapid enzymatic degradation, poor intracellular uptake, and excessively long base sequence to be synthesized. Considering the unique advantages of artificial nanomaterials in drug loading and transport, black phosphorus (BP) nanosheet was employed as a gene-drug carrier in this study. RESULTS: The sequence of PTENP1 was adopted as a template which was randomly divided into four segments with a length of about 1000 nucleotide bases to synthesize four different RNA fragments as gene drugs, and loaded onto polyethyleneimine (PEI)-modified BP nanosheets to construct BP-PEI@RNA delivery platforms. The RNAs could be effectively delivered into PC3 cells by BP-PEI nanosheets and elevating PTEN expression by competitive binding microRNAs (miRNAs) which target PTEN mRNA, ultimately exerting anti-tumor effects. CONCLUSIONS: Therefore, this study demonstrated that BP-PEI@RNAs is a promising gene therapeutic platform for PCa treatment.

Biodegradable FePS3 nanoplatform for efficient treatment of osteosarcoma by combination of gene and NIR-II photothermal therapy.

As a common tumor with high incidence, osteosarcoma possesses extremely poor prognosis and high mortality. Improving the survival of osteosarcoma patients is still a great challenge due to the precipice of advancement in treatment. In this study, a combination strategy of gene therapy and photothermal therapy (PTT) is developed for efficient treatment of osteosarcoma. Two-dimensional (2D) FePS3 nanosheets are synthesized and functionalized by poly-L-lysine-PEG-folic acid (PPF) to fabricate a multifunctional nanoplatform (FePS@PPF) for further loading microRNAs inhibitor, miR-19a inhibitor (anti-miR-19a). The photothermal conversion efficiency of FePS@PPF is up to 47.1% under irradiation by 1064 nm laser. In vitro study shows that anti-miR-19a can be efficiently internalized into osteosarcoma cells through the protection and delivery of FePS@PPF nanaocarrier, which induces up-regulation of PTEN protein and down-regulation p-AKT protein. After intravenous injection, the FePS@PPF nanoplatform specifically accumulates to tumor site of osteosarcoma-bearing mice. The in vitro and in vivo investigations reveal that the combined PTT-gene therapy displays most significant tumor ablation compared with monotherapy. More importantly, the good biodegradability promotes FePS@PPF to be cleared from body avoiding potential toxicity of long-term retention. Our work not only develops a combined strategy of NIR-II PTT and gene therapy mediated by anti-miR-19a/FePS@PPF but also provides insights into the design and applications of other nanotherapeutic platforms.

Mediated Drug Release from Nanovehicles by Black Phosphorus Quantum Dots for Efficient Therapy of Chronic Obstructive Pulmonary Disease.

Chronic obstructive pulmonary disease (COPD) is an intractable disease involving a sticky mucus layer and nanoagents with mucus-penetrating capability offer a new way to deliver drugs. However, drug release from nanovehicles requires optimization to enhance the therapeutic effects of COPD therapy. Herein, black phosphorus quantum dots (BPQDs) are combined with PEGylated chitosan nanospheres containing the antibiotic amikacin (termed PEG@CS/BPQDs-AM NPs). As a drug-delivery system, the hydrophilicity of PEG and positive charge of CS facilitate the penetration of nanovehicles through the mucus layer. The nanovehicles then adhere to the mucous membrane. Furthermore, the BPQDs degrade rapidly into nontoxic PO43- and acidic H+ , thereby promoting the dissociation of PEGylated CS nanospheres, accelerating the release of AM, decreasing the vitality of biofilms for ease of eradication. Our results reveal that drug delivery mediated by BPQDs is a feasible and desirable strategy for precision medicine and promising for the clinical therapy of COPD.

Two cholesterol derivative-based PEGylated liposomes as drug delivery system, study on pharmacokinetics and drug delivery to retina.

In this study, two cholesterol derivatives, (4-cholesterocarbonyl-4'-(N,N,N-triethylamine butyloxyl bromide) azobenzene (CAB) and 4-cholesterocarbonyl-4'-(N,N-diethylamine butyloxyl) azobenzene (ACB), one of which is positively charged while the other is neutral, were synthesized and incorporated with phospholipids and cholesterol to form doxorubicin (DOX)-loaded liposomes. PEGylation was achieved by including 1,2-distearoyl-sn-glycero-3-phosphatiylethanol-amine-N-[methoxy-(polyethylene glycol)-2000 (DSPE-PEG2000). Our results showed that PEGylated liposomes displayed significantly improved stability and the drug leakage was decreased compared to the non-PEGylated ones in vitro. The in vivo study with rats also revealed that the pharmacokinetics and circulation half-life of DOX were significantly improved when liposomes were PEGylated (p < 0.05). In particular, the neutral cholesterol derivative ACB played some role in improving liposomes' stability in systemic circulation compared to the conventional PC liposome and the positively charged CAB liposome, with or without PEGylation. In addition, in the case of local drug delivery, the positively charged PEG-liposome not only delivered much more of the drug into the rats' retinas (p < 0.001), but also maintained much longer drug retention time compared to the neutral PEGylated liposomes.

Transformation of Black Phosphorus through Lattice Reconstruction for NIR-II-Responsive Cancer Therapy.

The photothermal performance of black phosphorus (BP) in the near infrared (NIR)-II bio-window (1000-1500 nm) is low, which limits its biomedical applications. Herein, ultrasmall nickel phosphide quantum dots (Ni2 P QDs) are synthesized with BP quantum dots (BPQDs) as the template by topochemical transformation. The size of Ni2 P QDs is ≈3.5 nm, similar to that of BPQDs, whereas the absorption and photothermal conversion efficiency of Ni2 P QDs at 1064 nm (43.5%) are significantly improved compared with those of BPQDs. To facilitate in vivo applications, an Ni2 P QDs-based liposomal nano-platform (Ni2 P-DOX@Lipo-cRGD) is designed by incorporation of Ni2 P QDs and doxorubicin (DOX) into liposomal bilayers and the interior, respectively. The encapsulated DOX is responsively released from liposomes upon 1064-nm laser irradiation owing to the photothermal effect of Ni2 P QDs, and the drug release rate and amount are controlled by the light intensity and exposure time. In vivo, experiments show that Ni2 P-DOX@Lipo-cRGD has excellent tumor target capability and biocompatibility, as well as complete tumor ablation through the combination of photothermal therapy and chemotherapy. The work provides a new paradigm for the NIR-II transformation of nano-materials and may shed light on the construction of multifunctional nano-platforms for cancer treatment.

BiTiS3 bio-transducer with explosive on-demand generation of NO gas for synergetic cancer therapy.

Combined photothermal therapy and nitric oxide (NO)-mediated gas therapy has shown great potential as a cancer treatment. However, the on-demand release of NO at a high concentration presents a challenge owing to the lack of an ideal bio-transducer with a high loading capacity of NO donors and sufficient energy to induce NO release. Here, we present a new 2D BiTiS3 nanosheet that is synthesized, loaded with the NO donor (BNN6), and conjugated with PEG-iRGD to produce a multifunctional bio-transducer (BNN6-BiTiS3-iRGD) for the on-demand production of NO. The BiTiS3 nanosheets not only have a high loading capacity of NO donors (750%), but also exhibit a high photothermal conversion efficiency (59.5%) after irradiation by a 1064-nm laser at 0.5 W/cm2. As a result of the above advantages, the temporal-controllable generation of NO within a large dynamic range (from 0 to 344 µM) is achieved by adjusting power densities, which is among the highest efficiency values reported for NO generators so far. Moreover, the targeted accumulation of BNN6-BiTiS3-iRGD at tumor sites leads to spatial-controllable NO release. In vitro and in vivo assessments demonstrate synergistic NO gas therapy with mild photothermal therapy based on BNN6-BiTiS3-iRGD. Our work provides insights into the design and application of other 2D nanomaterial-based therapeutic platforms.

Effect of Bioactive Black Phosphorus Nanomaterials on Cancer-Associated Fibroblast Heterogeneity in Pancreatic Cancer.

Tumor-stromal interactions and stromal heterogeneity in the tumor microenvironment are critical factors that influence the progression, metastasis, and chemoresistance of pancreatic ductal adenocarcinoma (PDAC). Here, we used spatial transcriptome technology to profile the gene expression landscape of primary PDAC and liver metastatic PDAC after bioactive black phosphorus nanomaterial (bioactive BP) treatment using a murine model of PDAC (LSL-KrasG12D/+; LSL-Trp53R172H/+; and Pdx-1-Cre mice). Bioinformatic and biochemical analyses showed that bioactive BP contributes to the tumor-stromal interplay by suppressing cancer-associated fibroblast (CAF) activation. Our results showed that bioactive BP contributes to CAF heterogeneity by decreasing the amount of inflammatory CAFs and myofibroblastic CAFs, two CAF subpopulations. Our study demonstrates the influence of bioactive BP on tumor-stromal interactions and CAF heterogeneity and suggests bioactive BP as a potential PDAC treatment.

Black Phosphorous Aptamer-based Platform for Biomarker Detection.

Black phosphorus nanostructures (nano-BPs) mainly include BP nanosheets (BP NSs), BP quantum dots (BPQDs), and other nano-BPs-based particles at nanoscale. Firstly discovered in 2014, nano-BPs are one of the most popular nanomaterials. Different synthesis methods are discussed in short to understand the basic concepts and developments in synthesis. Exfoliated nano-BPs, i.e. nano-BPs possess high surface area, high photothermal conversion efficacy, excellent biocompatibility, high charge carrier mobility (~1000 cm-2V-1s-1), thermal conductivity of 86 Wm-1K-1; and these properties make it a highly potential candidate for fabrication of biosensing platform. These properties enable nano-BPs to be promising photothermal/drug delivery agents as well as in electrochemical data storage devices and sensing devices; and in super capacitors, photodetectors, photovoltaics and solar cells, LEDs, super-conductors, etc. Early diagnosis is very critical in the health sector scenarios. This review attempts to highlight the attempts made towards attaining stable BP, BP-aptamer conjugates for successful biosensing applications. BP-aptamer- based platforms are reviewed to highlight the significance of BP in detecting biological and physiological markers of cardiovascular diseases and cancer; to be useful in disease diagnosis and management.

Combined chemotherapy based on bioactive black phosphorus for pancreatic cancer therapy.

Pancreatic cancer is the most aggressive malignant tumor with difficulty in early diagnosis, very short survival time in advanced stage, and lack of effective treatment options. In this work, a novel combination chemotherapy strategy based on bioactive black phosphorus (BP) and gemcitabine (GEM) is developed for efficient treatment of pancreatic cancer. The combined cell cycle blockage in G2/M phase induced by BP and G0/G1 phase by GEM results in synergistic killing of pancreatic cancer cells with the combination index (CI) < 1. The iRGD modified zein nanoparticles co-loaded with BP quantum dots (BPQDs) and GEM are designed and prepared as a targeted nanoplatform (BP-GEM@NPs). After intravenous injection, the in vivo distribution and pharmacokinetics results demonstrate that BP-GEM@NPs shows excellent tumor targeting capability and significantly prolonged blood circulation time. The targeted co-delivery of BPQDs and GEM induces much more pancreatic tumor cell apoptosis and synergistically inhibits tumor growth in both subcutaneous xenograft and orthotopic models. Meanwhile, BP-GEM@NPs exhibit good biocompatibility without bring adverse effects. This work indicates the great potential of BP-GEM@NPs as a combination chemotherapy for pancreatic cancer and provides insights into development of biomedicine by exploring the intrinsic bioactivities of nanomaterials.

Microencapsulation of Lead-Halide Perovskites in an Oil-in-Fluorine Emulsion for Cell Imaging.

The superior optical properties of lead-halide perovskites (LHPs) inspired significant research in cell imaging applications; many encapsulating processes have improved perovskite stabilities with comparable biosafety. Herein, facile solvent evaporation encapsulation based on an oil-in-fluorine emulsion for aqueous-stable and extremely nontoxic LHP microcapsules is described. Perfluorooctane dispersed the emulsifier fluorocarbon surfactant to form a continuous fluorine phase, while LHPs and polymethylmethacrylate (PMMA) were dispersed in 1,2-dichloroethane, then emulsified in the fluorine phase to form an oil-in-fluorine emulsion. CsPbBr3 microcapsules with a dense PMMA shell that protect fragile CsPbBr3 from the external environment and inhibit lead ion release were obtained after solvent evaporation. The CsPbBr3 microcapsules not only retained 91% of fluorescence intensity after exposure to water for 30 d but also possess extremely low cytotoxicity for MCF-7 cells. After exposure to 2 mg/mL of CsPbBr3 microcapsules for 48 h, the cell viability remained >90%. The intracellular uptake of CsPbBr3 microcapsules indicates its potential use in cell imaging.

Synthesis of germanium/germanium phosphide in-plane heterostructure with efficient photothermal and enhanced photodynamic effects in the second near-infrared biowindow.

Inspired by the bifunctional phototherapy agents (PTAs), constructing compact PTAs with efficient photothermal therapy (PTT) and photodynamic therapy (PDT) effects in the near-infrared (NIR-II) biowindow is crucial for high therapeutic efficacy. Herein, none-layered germanium (Ge) is transformed to layered Ge/germanium phosphide (Ge/GeP) structure, and a novel two-dimensional sheet-like compact S-scheme Ge/GeP in-plane heterostructure with a large extinction coefficient of 15.66 L/g cm-1 at 1,064 nm is designed and demonstrated. In addition to the outstanding photothermal effects, biocompatibility and degradability, type I and type II PDT effects are activated by a single laser. Furthermore, enhanced reactive oxygen species generation under longer wavelength NIR laser irradiation is achieved, and production of singlet oxygen and superoxide radical upon 1,064 nm laser irradiation is more than double that under 660 nm laser irradiation. The S-scheme charge transfer mechanism between Ge and GeP, is demonstrated by photo-irradiated Kelvin probe force microscopy and electron spin resonance analysis. Thus, the obtained S-scheme Ge/GeP in-plane heterostructure shows synergistic therapeutic effects of PTT/PDT both in vitro and in vivo in the NIR-II biowindow and the novel nanoplatform with excellent properties has large clinical potential.

Colorimetric immunosensing using liposome encapsulated MnO2 nanozymes for SARS-CoV-2 antigen detection.

Development of specific signal reporters with signal amplification effect are highly needed for sensitive and accurate detection of pathogen. Herein, we design a colorimetric immunosensing nanosystem based on liposome encapsulated quantum dots-sized MnO2 nanozyme (MnO2QDs@Lip) as a signal reporter for ultrasensitive and fast detection of SARS-CoV-2 antigen. The pathogenic antigens captured and separated by antibody-conjugated magnetic beads (MBs) are further connected with antibody-modified MnO2QDs@Lip to form a sandwich-like immunocomplex structure. After triggered release, MnO2 QDs efficiently catalyze colorless 3,3',5,5'-tetramethylbenzidine (TMB) to blue oxidized TMB, which can be qualitatively observed by naked eyes and quantitatively analyzed by UV-Vis spectra or smartphone platforms. By taking advantages of immuno-magnetic separation, excellent peroxidase-like catalytic activity of MnO2 QDs, and high encapsulation efficiency of MnO2QDs@Lip, ultrasensitive detection of SARS-CoV-2 antigen ranging from 0.1 pg/mL to 100 ng/mL is achieved within 20 min. The limit of detection (LOD) is calculated to be 65 fg/mL in PBS buffer. Furthermore, real clinical samples of SARS-CoV-2 antigens can be effectively identified by this immunosensing nanosystem with excellent accuracy. This proposed detection nanosystem provides a strategy for simple, rapid and ultrasensitive detection of pathogens and may shed light on the development of new POCT detection platforms for early diagnosis of pathogens and surveillance in public health.

Niobium Diselenide Nanosheets: An Emerging Biodegradable Nanoplatform for Efficient Cancer Phototheranostics in the NIR-II Window.

As a prominent class of 2D transition metal dichalcogenides (TMDCs), niobium diselenide nanosheets (NbSe2 NSs) have garnered tremendous interest on account of promising applications pertaining to optoelectronics and energy storage. Although NbSe2 NSs have many unique advantages such as inherent biocompatibility and broad absorption in the NIR region, their biomedical applications have rarely been reported, especially as therapeutic agents for the second near-infrared (NIR-II) range. Herein, a biodegradable nanotherapeutic platform consisting of NbSe2 NSs is designed and demonstrated for NIR-II light-triggered photothermal therapy. NbSe2 NSs synthesized by grinding and liquid exfoliation exhibit superior photothermal conversion efficiency (48.3%) and remarkable photothermal stability in the NIR-II region. In vitro assessment demonstrates that NbSe2 NSs have favorable photothermal cell ablation efficiency and biocompatibility. After intravenous injection in vivo, the NbSe2 NSs accumulate passively in tumor sites to facilitate fluorescence imaging and tumor ablation by NIR-II illumination. Furthermore, as a result of gradual degradation in the physiological environment, NbSe2 NSs can be excreted from the body to avoid potential toxicity caused by long-term retention in vivo. The results reveal a promising NIR-II light-triggered PTT strategy with the aid of NbSe2 NSs and the platform is expected to have large potential in cancer theranostics.

Intrinsic bioactivity of black phosphorus nanomaterials on mitotic centrosome destabilization through suppression of PLK1 kinase.

Although nanomaterials have shown promising biomedical application potential, incomplete understanding of their molecular interactions with biological systems prevents their inclusion into mainstream clinical applications. Here we show that black phosphorus (BP) nanomaterials directly affect the cell cycle's centrosome machinery. BP destabilizes mitotic centrosomes by attenuating the cohesion of pericentriolar material and consequently leads to centrosome fragmentation within mitosis. As a result, BP-treated cells exhibit multipolar spindles and mitotic delay, and ultimately undergo apoptosis. Mechanistically, BP compromises centrosome integrity by deactivating the centrosome kinase polo-like kinase 1 (PLK1). BP directly binds to PLK1, inducing its aggregation, decreasing its cytosolic mobility and eventually restricting its recruitment to centrosomes for activation. With this mechanism, BP nanomaterials show great anticancer potential in tumour xenografted mice. Together, our study reveals a molecular mechanism for the tumoricidal properties of BP and proposes a direction for biomedical application of nanomaterials by exploring their intrinsic bioactivities.

Bioactive phospho-therapy with black phosphorus for in vivo tumor suppression.

Background and Purpose: Although inorganic nanomaterials have been widely used in multimodal cancer therapies, the intrinsic contributions of the materials are not well understood and sometimes underestimated. In this work, bioactive phospho-therapy with black phosphorus nanosheets (BPs) for in vivo tumor suppression is studied. Methods: Orthotopic liver tumor and acute myeloid leukemia are chosen as the models for the solid tumor and hematological tumor, respectively. BPs are injected into mice through the tail vein and tumor growth is monitored by IVIS bioluminescence imaging. Tumor tissues and serum samples are collected to determine the suppression effect and biosafety of BPs after treatment. Results: The in vitro studies show that BPs with high intracellular uptake produce apoptosis- and autophagy-mediated programmed cell death of human liver carcinoma cells but do not affect normal cells. BPs passively accumulate in the tumor site at a high concentration and inhibit tumor growth. The tumor weight is much less than that observed from the doxorubicin (DOX)-treated group. The average survival time is extended by at least two months and the survival rate is 100% after 120 days. Western bolt analysis confirms that BPs suppress carcinoma growth via the apoptosis and autophagy pathways. In addition, administration of BPs into mice suffering from leukemia results in tumor suppression and long survival. Conclusions: This study reveals that BPs constitute a type of bioactive anti-cancer agents and provides insights into the application of inorganic nanomaterials to cancer therapy.

InSe Nanosheets for Efficient NIR-II-Responsive Drug Release.

Near-infrared-II (NIR-II) biowindow is appealing from the perspectives of larger maximum permissible exposure in comparison with the near-infrared-I biowindow, so the NIR-II-responsive drug-delivery nanoplatform is highly desirable. In this work, two-dimensional InSe nanosheets (InSe NSs) are modified with poly(ethylene glycol) and evaluated as an effective NIR-II-responsive cancer treatment nanoplatform. The InSe NSs synthesized by liquid exfoliation exhibit prominent NIR-II-responsive photothermal conversion efficiency (39.5%) and photothermal stability. Moreover, the InSe NSs have a doxorubicin (DOX) loading capacity as high as 93.6%, along with excellent NIR-II-responsive DOX release characteristic. The superior synergistic chemo/photothermal effects have also been demonstrated by the in vitro experiments in killing cancer cells. In combination with good biocompatibility, the InSe NSs have great potential in therapeutic applications.

Synthesis of lipid-black phosphorus quantum dot bilayer vesicles for near-infrared-controlled drug release.

Black phosphorus quantum dots are incorporated into liposomal bilayers to produce a drug delivery system with excellent near-infrared (NIR) photothermal properties and drug release capability controlled by light. In vitro experiments demonstrate its good biocompatibility and NIR-light-induced chemo-photothermal antitumor efficiency.

A Light-Responsive Self-Assembly Formed by a Cationic Azobenzene Derivative and SDS as a Drug Delivery System.

The structure of a self-assembly formed from a cationic azobenzene derivative, 4-cholesterocarbonyl-4'-(N,N,N-triethylamine butyloxyl bromide) azobenzene (CAB) and surfactant sodium dodecyl sulfate (SDS) in aqueous solution was studied by cryo-TEM and synchrotron radiation small-angle X-ray scattering (SAXS). Both unilamellar and multilamellar vesicles could be observed. CAB in vesicles were capable to undergo reversible trans-to-cis isomerization upon UV or visible light irradiation. The structural change upon UV light irradiation could be catched by SAXS, which demonstrated that the interlamellar spacing of the cis-multilamellar vesicles increased by 0.2-0.3 nm. Based on this microstructural change, the release of rhodamine B (RhB) and doxorubicin (DOX) could be triggered by UV irradiation. When incubated NIH 3T3 cells and Bel 7402 cells with DOX-loaded CAB/SDS vesicles, UV irradiation induced DOX release decreased the viability of both cell lines significantly compared with the non-irradiated cells. The in vitro experiment indicated that CAB/SDS vesicles had high efficiency to deliver loaded molecules into cells. The in vivo experiment showed that CAB/SDS vesicles not only have high drug delivery efficiency into rat retinas, but also could maintain high drug concentration for a longer time. CAB/SDS catanionic vesicles may find potential applications as a smart drug delivery system for controlled release by light.

Physical stability of cholesterol derivatives combined with liposomes and their in vitro behavior.

The purpose of this study was to investigate the physical stability and drug release of two cholesterol derivatives (4-cholesterocarbonyl-4'-(N,N,N-triethylamine butyloxyl bromide, CTBBA, and 4-cholesterocarbonyl-4'-(N,N'-diethylamino-butyloxy, CDBA), when combined with doxorubicin (DOX)-loaded liposomes in vitro. CTBBA-liposome revealed a positive charge at a pH between 3 and 10, as indicated by the ζ-potential. DOX-encapsulated CTBBA-liposomes possessed better physical stability both in PBS and in fetal bovine serum (FBS) added to PBS.

Preparation of zein conjugated quantum dots and their in vivo transdermal delivery capacity through nude mouse skin.

Zein have been successfully conjugated with quantum dots (QD630) via N-(3-dimethylaminopropyl)-N(-ethyl-carbodiimide hydrochloride (EDC) coupling. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) measurements indicated that the average diameter of QD630 was about 5 nm, and that of the zein conjugated QD630 (QD630/Zein) increased to 24 nm. Their size distributions were relatively narrow (Polyindex (PI) < 0.15). The FT-IR and electrophoresis results indicated that QD630 has been combined with zein covalently. Uptakes of QD630 and QD630/Zein by NIH3T3 cells showed different kinetics as observed by fluorescent microscope. There was no obvious increase of fluorescent intensity within cells after 1 h for QD630 sample, while for QD630/Zein, uptake was found to be continuously increased and the major portion of QD630/Zein was localized in the perinuclear area in the form of granular inclusions at 9 h and 24 h. Moreover, penetrating ability of QD630 and QD630/Zein into skin was evaluated with nude mouse in vivo. Fluorescence images revealed that tiny QD630 particles penetrated through the stratum corneum barrier and localized within the epidermal and dermal layers by 8 h, and no QDs was found after 24 h; whereas for QD630/Zein particles, no obvious penetration was observed within 24 hours. Modification of conjugated zein to QDs may alter their skin penetration characteristics and reduce the risk of the QD toxicity.