NIR Emissive Biomimetic Ghost Nanovesicles for Site-Selective Solid Tumor Imaging

NIR Emissive Biomimetic Ghost Nanovesicles for Site-Selective Solid Tumor Imaging | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article NIR Emissive Biomimetic Ghost Nanovesicles for Site-Selective Solid Tumor Imaging RAJENDRA PRASAD, Kumari Prerna, Mayur Temgire, Pinaki Banerjee, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4616433/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 3 You are reading this latest preprint version Abstract Optically active biomimetic ghosts nanovesicles are highly potent as imaging agents for site-selective solid tumor imaging with deep tissue visualization. However, reported systems are limited with poor brightness and photostability with NIR absorption and emission. Herein, cancer cell membrane derived biomimetic ghost nanovesicles (~60 nm) have been engineered with amphiphilic dyes aggregates for site-selective solid tumor imaging in pre-clinical models. Entrapped dye aggregates within biomimetic ghost nanovesicles (BNVs, 505 to 828 dye molecules/vesicle) exhibit promising fluorescence and photostability (up to 30 days) showing ultra-brightness (778 MESF) with promising tumor fluorescence signals (760 nm excitation) compared to free dye molecules and dye aggregates. Dye aggregates-BNVs exhibit significantly different imaging response than amphiphilic monomers-BNVs. Lipophilic and amphiphilic structural layers and surface biomarkers of ghost nanovesicles are examined through physicochemical measurements, corroborated with cargo release kinetics. Controlled body weight, long time survival and histopathology examinations ensure the in vivo biocompatibility of these intravenously administrated biomimetic imaging agents. Our findings suggest that these ghosts nanovesicles mimic the biological characteristics of native cells, enabling them to evade immune clearance. Biological sciences/Biotechnology Physical sciences/Nanoscience and technology Physical sciences/Optics and photonics Biomimetic Ultrabright Nanoparticles In vivo Imaging Solid Tumor Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction N ear-infrared responsive fluorescence (NIRF) imaging has been considered as versatile and safe approach for visualizing abnormal tissues/tumor selectively in pre-clinical and clinical models. 1–3 Among non-invasive contrast agents, optically active imaging probes offer several advantages for site-selective diagnosis of solid tumors, assessing the blood perfusion, real-time cancer lymphography, etc., due to deep tissue penetration, low light scattering, and high spatial resolution of the near-infrared light. 4–8 Moreover, NIR emissive nanoprobes exhibit low photon absorption and light scattering, resulting in a high signal-to-noise ratio with promising diagnostic ability. 9–14 Indocyanine green (ICG, NIR fluorescent dye) 9 is one such imaging probe approved by the U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA) for pre-clinical and clinical applications. 15 , 16 Overall, ICG dye has been recognized as a promising contrast agent with low interference from tissue autofluorescence and blood absorption (500-600 nm) due to absorption and emission in the NIR spectral range (650-900 nm). 17 Apart from cancer diagnosis, ICG dye has been applied to examine the various cardiac, liver, and ophthalmic angiography functions due to enhanced fluorescence intensity of plasma-integrated ICG aggregates 18–21 To the best of our knowledge and based on the literature survey, we can note that the fluorescence intensity and lifetime may fluctuate with varying plasma concentrations (protein and lipoprotein). 14,15 Nonspecific biodistribution, low blood circulation time, and photodegradation of free organic ICG dye are major limitations in medical applications. 22 These amphiphilic ICG molecules show molecular aggregation in the aqueous environments, resulting in fluorescence quenching 23 , 24 ,which diminishes/reduces their fluorescence and light mediated therapeutics ability. Recently, aggregation-induced emission (AIE) 25 , 26 has been considered a new development in fluorophores, which resolves the problem of aggregation-induced quenching of organic imaging probes. Particularly, J-aggregates of organic dyes (slip-stacked alignment) 27 have been used for bioimaging due to delocalized π-electrons, which leads to constructive coupling of the excited state transition dipoles (Figure 1A-D). In spectroscopic manner, organic dye aggregates (J-aggregates) produce bathochromic shift in absorption and emission with narrow bands, enhanced molar extinction coefficient (ε), and shortened fluorescence lifetimes with better quantum yields (ΦF) which decide their average brightness. 28–31 Aggregates of organic dyes like squaraine, flavylium, porphyrin, perylene bisimide derivatives, heptamethine cyanine, and pyrrolopyrrole cyanine have been reported but limited with low photostability, easy degradation, nonspecific biodistribution and binding which limit their ability for site-selective tumor imaging. 31–36 Therefore, encapsulating these emissive molecules in the nanoparticles can resolve the aforementioned obstacles. For example, in 2016, aggregate porphyrin-lipids nanoparticles were tested for image-guided surgery. 37 Recently, squaraine J-aggregate-polymeric micelles have been tested for image-guided photothermal therapy. 35 However, premature leakage of loaded emissive molecules, poor brightness, low colloidal stability, and lack of specific, and natural tumor targeting ability are remaining challenges of these nanosized formulations. 38–40 Herein, we report cancer cell derived biomimetic ghost nanovesicles entrapping amphiphilic dye aggregates (ICG and Nile Red) within bilayers, internal cavity, and exterior surface, demonstrating their structural characteristics. Further, these engineered optically active biomimetic ghost nanovesicles were tested for solid tumor imaging in pre-clinicalmodels. For these optically active biomimetic imaging agents, aqueous solution of dye (0.01 mM in the aqueous medium) has been used to prepare fluorescent amphiphilic dye aggregates whereas monomers of dye have been tested as photosensitizer. RESULTS AND DISCUSSION Characteristics of dye tagged biomimetic nano ghost vesicles are evaluated through microscopic and spectroscopic measurements which are further corroborated by in vitro and in vivo studies (Fig. 2 A-E). Dye integrated (J-aggregates) ghost nanovesicles (~ 60 nm) show NIR (650–900 nm) absorption and emission which have been tested for solid tumor imaging and localized biodistribution. Engineered optically active biomimetic imaging agents 41 are stable for several days (tested up to 30 days, see supporting information for more details) showing promising brightness (779 MESF for 784 ICG/BNV and 2.69 × 10 11 BNVs/cm 3 , Fig. 3 and Table 1) for localized tumor visualization. Moreover, phototransduction response of designed biomimetic optically active ghost nanovesicles (J-aggregates and monomers of ICG dye entrapped nanovesicles) have been evaluated due to their NIR absorption ability. Upon 5 minutes of NIR light exposure, J-aggregates of ICG dye demonstrate better phototransduction (57°C at 2.5×10 13 nanoparticles/cm 3 ) compared to ICG monomers (44°C at 2.5×10 13 nanoparticles/cm 3 ). Whereas ICG monomers tagged biomimetic nanovesicles exhibit better photosensitizing property as compared to J-aggregates encapsulated nanovesicles (details are discussed below). On the other hand, we have noticed that ICG J-aggregates encapsulated nanovesicles exhibit better brightness (754 MESF units) than their monomer (598 MESF units) may be due to self-quenching of free monomers in nanovesicles. Additionally, it is believed that monomers of small organic dye molecules can diffuse easily from the interior of nanovesicles as compared to their aggregated form. Hence, they may exhibit poor emission property which may not be suitable for bioimaging applications. It should be noted that nanovesicles have lipid assemblies in their membrane. Hence, lipid specific dye remains in the membrane for a long time (at least 12–24 h) due to lipophilic nature of small organic molecules. In the present work, we have used Nile Red, which is specific to the lipid layers with BNVs, but they require high numbers of dye molecules per particle (802) and a number of BNVs per cm 3 (7.90 × 10 12 ) to exhibit comparable brightness (748 MESF units) to the ICG J-aggregates in BNVs due to molar extinction coefficient (ε, 38000 cm - 1 M - 1 ) barriers. Cancer cell membrane ghost vesicles 42 , 43 (3–5 mg/mL) are prepared from breast cancer cells (5×10 5 -7×10 5 cancer cells/experiments) in hypotonic conditions treated with various buffers (ice-cold Tris-Magnesium and PBS) followed by freeze-thaw and centrifugal process (Supplementary Fig. 1A). These ghost vesicles are used to prepare nanosized biomimetic cell membrane vesicles under multiple rounds of probe sonication (20 cycles) in ice bath. Uniform size distribution (~ 60 nm, Supplementary Fig. 1B) of prepared biomimetic cell membrane vesicles has been examined by cryo-mode TEM, which is corroborated by dynamic light scattering measurements (0.28 PDI) (Supplementary Fig. 1C). Diameter sharpness in light scattering measurements indicates nanovesicles' spherical morphology, which is clearly ensured by microscopic imaging. On the other hand, the negative surface charge (-12 mV) of nanovesicles supports their colloidal stability in physiological conditions, which may be due to the presence of natural surface biomarkers (Supplementary Fig. 1D). These available inherent surface biomarkers, mostly proteins, help in (i) better particle dispersion in the physiological environment observed from microscopic images shown in Supplementary Fig. 1E, (ii) natural targeting, and (iii) evading macrophage uptake (data are not shown here). Interestingly, controlled particle diameter (~ 68 nm) in the aqueous medium indicated the better dispersion of nanovesicles over 30 days of storage (Supplementary Fig. 1E and Supplementary Fig. 2A). More importantly, we are trying to address major hurdles such as (i) are these cells derived nanovesicles exhibit homogeneous size distribution and (ii) what is the fate of repeatability and scalability of these biomimetic nanovesicles? Secondly, we also have tried to understand the numbers of encapsulated dye molecules which decides the brightness of particles. To address these critical challenges, we have optimized the preparation recipe and made sure the reproducibility for more than 10 times. However, we have noticed slight changes in the final product concentration (1.20 to 1.32 mg/mL), particles size (60 to 65 nm) distribution and numbers of dye molecules per nanovesicle (201 to 281) during each batch (see Supplementary Table 1 and Table 2 in the supporting information). One the other hand, batch scalability is also a major challenge especially for liposomes and cell derived biomimetic nanovesicles which has been specifically taken care while optimizing the present recipe. We have noticed that concentration of magnesium chloride and sucrose in Tris buffer and Freeze-Thaw time are essential components apart from mechanical forces which decide the cell ghost structures and surface biomarkers. Results of 10 different batches demonstrate the consistency in final product concentration (1.20 to 1.32 mg/mL concentration per 15 mL batch). It should be noted that the biomimetic ghost nanovesicles have been achieved with 5 mg/mL maximum yield but that was for large scale production (100 mL volume of cell suspension) where 65 nm size of nanovesicle can encapsulate 215 ICG dye molecules in its cavity. $$Weightof 1 BNV \left(mg\right)=\rho \times 4/3\pi r^3 \left(1\right)$$ $$NumberofBNVs/mL=C/(\rho \times 4/3\pi r^3) \left(2\right)$$ $$NumberofDyes/mL=A/ \epsilon \times 6.023\times 10^23 \left(3\right)$$ So far, several studies have been reported on cell membrane-derived biomimetic nanovesicles and their integration with fluorescent dye molecules. 14 However, ultra brightness and the importance of integrated dye for the structural characteristic of membrane nanovesicles have yet to be achieved. In the current study, we show the amphiphilic dye molecules (ICG and Nile Red) integrated biomimetic ghost nanovesicles at the nanoscale for targeted tumor imaging and specific biodistribution. In microscopic images, observed dark patches from the (i) cavity (505 to 784 ICG molecules/nanovesicle), (ii) exterior surface (578 to 828 ICG molecules/nanovesicle), and (iii) bilayer of single nanovesicle represent the assembly of amphiphilic dye molecules (ICG and Nile Red) in the form of J-aggregates (Fig. 2 A). Details of nanovesicle numbers, dye per nanovesicles and respective brightness are thoroughly discussed in the supporting information (Supplementary Table 1). These observations are further corroborated with spectroscopic measurements, as shown in Fig. 2 B, C. In absorption spectra, aqueous ICG monomers (0.01 mM) exhibit λ max absorbance at 780 nm and a weaker H-aggregate peak at 710 nm. Aqueous dye at high temperature (60°C) incubation result in miniature J-aggregates showing bathochromic shifts in absorption and emission (λ abs/emm 890/804 nm) with peak sharpness. Further, these J-aggregates exhibit significant bathochromic shifts in absorption and emission (λ abs/emm 893/806 nm) upon their integration with biomimetic cell ghost nanovesicles which are purified through dialysis (for 2 days using 12 KD dialysis membrane). Photo stability of dye tagged nanovesicles have been evaluated by measuring their emission properties at various time points (1 h to 30 days) with and without treating in serum (Supplementary Fig. 2B, C). It has been calculated that a single membrane ghost nanovesicles accommodate a few hundred dye molecules exhibiting ultra brightness (498 to 778 MESF units viz., Molecules of Equivalent Soluble Fluorophore). Controlled emission and maintained brightness for a prolonged period (tested up to 30 days) demonstrate the photostability of emissive ghost nanovesicles, whereas free dye J-aggregates demonstrate ~ 40% degradation and reduced emission (Supplementary Fig. 2A-C). Overall, the formed J-aggregates demonstrate better fluorescence intensity and stability under 760 nm excitation when they are within the nanoparticulate formulation. It should be noted that free ICG in aqueous solution mainly exhibit their monomers with two absorption peaks at 780 and 712 nm, respectively whereas these monomers turn into aggregated form (J aggregates, a sharp extinction peak at 890 nm) due to noncovalent π–π stacking and hydrophobic interactions upon 60°C incubation for 2 h. Further, these ICG J-aggregates integrated ghost nanovesicles exhibit hyperthermia temperature (43°C at 2.5×10 13 nanoparticles/cm 3 ) within 2 minutes which is further improved to 57°C after 5 minutes of NIR exposure compared to ICG monomers encapsulated nanovesicles (44.6°C at 2.5×10 13 nanoparticles/cm 3 ) as shown in Supplementary Fig. 2D. Whereas ICG monomers tagged biomimetic ghost nanovesicles exhibit better photosensitizing property (80–85%) as compared to J-aggregates encapsulated nanovesicles (20%, see Supplementary Fig. 2E) which indicate their photodynamic therapeutics applicability (data not shown here). Next, microscopic characteristics reveal that amphiphilic ICG J-aggregates integrated nanovesicles exhibit the exterior dark layer (5–6 nm) around due to the presence of aggregated dye molecules within the available surface biomarkers of nanovesicles which is corroborated by spectroscopic measurements (λ max , absorbance at 892–893 nm and λ max , emission at 806 nm) as shown in Fig. 2 B, C. Similarly, the presence of lipid bilayers (2–3 nm) within biomimetic nanovesicles is physiochemically characterized. J-aggregates of lipophilic Nile Red dye in nanovesicles show bathochromic shifts (34 nm red shift) with λ max , absorbance at 604 nm, and emission at 662 nm as compared to their monomers (λ max , absorbance at 570 nm, Supplementary Fig. 3) which is also supported by microscopic characterization. The presence of amphiphilic J-aggregates in nanovesicles is validated through release kinetic patterns in cancer mimicked conditions viz., acidic conditions (Fig. 2 D). Dye from the exterior surface of vesicles show rapid release as compared to J-aggregates inside the vesicles (Supplementary Fig. 4 and Supplementary Fig. 5). These nanovesicles demonstrate better cargo release (more than 10%) in late endosomal (pH 2–4) and tumor mimicked environment (pH 6.5) as compared to physiological environment (less than 10% at pH 7.4). Further, the red patches from cancer cells (MDA-MB231 and 4T1) interior indicate the presence of red emissive dye aggregates (ICG and Nile Red) within the BNVs located at different nanovesicle sites. Inherent surface biomarkers of nanovesicles exhibit strong binding with breast cancer cells (MDA-MB231), indicating their natural targeting ability shown in Fig. 2 E, Fig. 3 , and Supplementary Fig. 6. Empty ghost nanovesicles show distinct cavities as compared to J-aggregates integrated nanovesicles confirmed through microscopic characterization (Fig. 2 A, Supplementary Fig. 1B, and Supplementary Fig. 7). These characteristics are discussed here for the first time in the case of cell membrane-derived biomimetic imaging agents. $$\text{R}\text{e}\text{l}\text{a}\text{t}\text{i}\text{v}\text{e} \text{b}\text{r}\text{i}\text{g}\text{h}\text{t}\text{n}\text{e}\text{s}\text{s}=\frac{\frac{\text{F}\text{L}\text{B}\text{N}\text{V}}{C\text{B}\text{N}\text{V}}}{\frac{\text{F}\text{L}\text{D}\text{y}\text{e}}{C\text{D}\text{y}\text{e}}} \left(4\right)$$ Further, these bright nanovesicles have also been tested for targeted cancer cell imaging (4T1 metastatic breast cancer cells), as shown in Fig. 3 A. After 3 h of post-treatment, red emissive dye-tagged nanovesicles are accumulated in cancer cells. It is observed that the brightness of dye-tagged nanovesicles is dependent on the number of dyes per nanovesicle, which define as a multiplication of the quantum yields (ΦF) and the extinction coefficient ε(λ) (M − 1 cm − 1 units) (Fig. 3 B and equations 1–4). In the present work, the brightness of nanoparticles is calculated by considering the 2.621×10 5 M − 1 cm − 1 extinction coefficient at various concentrations of nanoparticles (0.9 to 2.5 mg/mL, Supplementary Table 1). The prepared ultrabright nanovesicles are tested for in vitro and in vivo biocompatibility. In vitro compatibility, BNVs before and after different cargo (J-aggregates of ICG and Nile Red) loading show more than 95% cell viability (200 µg/mL) of healthy cells which is recorded 80–85% at 500 µg/mL (Fig. 3 C). It should be noted that engineered BNVs demonstrate multifunctional abilities due to entrapped different cargo molecules (ICG, and Nile Red), which are examined through physicochemical studies and release kinetics (negligible release is seen at neutral pH 7.4), indicating safe entrapment of loaded cargo molecules (Fig. 2 , Supplementary Fig. 8 and Supplementary Fig. 9). Brightness and contrast response of J-aggregates (ICG and Nile Red) tagged BNVs have been evaluated in tumor-bearing female mice at different concentrations (0.1-1 mg/mL). Remarkably, ICG J-aggregates-tagged BNVs demonstrate 20-fold brightness compared to only ICG J-aggregates in the aqueous medium as shown in Supplementary Fig. 10. To evaluate the site-selective tumor imaging and specific biodistribution, a single dose (2.5×10 13 nanoparticles/cm 3 ) of J-aggregates (ICG and Nile Red) tagged BNVs are intravenously administrated to 4T1 tumor-bearing female Balb/c mice. After post-injection, whole-body scans are captured through in vivo imaging system at different time points (0.5 h to 24 h), indicating a clear visualization of the tumor site with better brightness signal intensity (Fig. 4 A-C). In initial post-injection time (0.5 h to 1 h) the injected ultrabright BNVs contrast agents adjust their distribution (0.98×10 8 to 0.23×10 8 p/sec/cm 2 /sr) among vital organs (heart, liver, spleen) and increase in the tumor site (0.065×10 8 to 1.98×10 8 p/sec/cm 2 /sr) from 3 h of post-injection time which is further constant (2.2×10 8 to 2.6×10 8 p/sec/cm 2 /sr) up to 24 h of post-injection (Fig. 4 A-C and Supplementary Fig. 11 and Supplementary Fig. 12). On the other hand, it has been noted that ICG J-aggregates-BNVs (exterior) exhibit higher signal intensity and brightness (2.4×10 8 p/sec/cm 2 /sr) from the tumor site as compared to ICG J-aggregates-BNVs (interior) (2.0×10 8 p/sec/cm 2 /sr) indicating the quick release response of emissive dye from exterior surface of BNVs in the cancer mimicked environment as shown in Fig. 4 A. Interestingly, we have noticed that injected ultrabright BNVs contrast agents identify the metastasis in tumor bearing mice. Moreover, Nile Red J-aggregates-BNVs also demonstrate promising signal intensity (1.4×10 8 p/sec/cm 2 /sr) from the tumor site which augment (1.6×10 8 to 2.6×10 8 p/sec/cm 2 /sr) with post-injection time (3 h to 24 h). Controlled and uniform fluorescence from the tumor area indicates the better accumulation and distribution of injected red emissive biomimetic nanovesicles than the pre-injected mice (Supplementary Fig. 12 and Supplementary Fig. 13). Biodistribution measurements have been evaluated by considering quantitative ROI values of the fluorescence intensity of the corresponding organs and tumors in vivo (Fig. 4 B, C). Further, the promising brightness and fluorescence from the whole body demonstrate the better circulation of nanovesicles, which are entering and stay within solid tumors and exiting via draining tumor lymphatic and then re-entering in the blood circulation as shown in Fig. 4 . However, the clear mechanism is not known yet. Next, these injected nanovesicles are site-specific for solid tumor without affecting healthy tissues (heart, lung, liver, spleen and kidneys) showing their better biocompatibility due to their natural surface biomarkers and biomimetic ability. The tissue toxicity has been evaluated through histopathology examinations as shown in Fig. 4 D. Tissue histology exhibits no pathological damage or changes to the vital organs where (i) myofiber and muscle bundle in the heart, (ii) portal triad and central vein in the liver are noticed without any injury. Moreover, the glomerulus and tubules in the kidney are also without any histological change. On account of unique features, we can state that the engineered biomimetic ultrabright nanovesicles are safe to be used as contrast agents for localized tumor imaging and tissue visualization in vivo . CONCLUSION In summary, we present the safe bioengineering of cancer cell membrane derived biomimetic ghost nanovesicles which are tagged with amphiphilic dyes (ICG and Nile Red) aggregates for site-selective solid tumor imaging in pre-clinical models. Structural characteristics of these engineered biomimetic nanovesicles has been examined through microscopic and spectroscopic methods. These dye entrapped ghost nanovesicles exhibit intense brightness and signals intensity along with better phototransduction and photosensitizing ability. During diagnostics course, the primary tumor location and tumor metastasis have been clearly observed. Maintained body weight, long time survival and histopathology examinations ensure the in vivo biocompatibility of these intravenously administrated biomimetic imaging agents. Among several reports, the structural features of engineered biomimetic nanovesicles have been discussed here, which are correlated with various physicochemical, in vitro , and in vivo studies. Hence, the developed cancer cell membrane vesicles could be considered clinically relevant imaging and therapeutics medicine. EXPERIMENTAL SECTION Materials and techniques . Indocyanine green (ICG), Nile Red (NR), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) were purchased from Sigma-Aldrich. Dulbecco's Modified Eagle Medium (DMEM), Phosphate buffered saline (PBS, pH 7.4), dimethyl sulphoxide (DMSO), Fetal bovine serum (FBS), 4’,6-diamidino-2-phenylindole (DAPI) and antibiotic-antimycotic solution were obtained from HiMedia Pvt. Ltd. Milli-Q (> 18.2 MΩ cm) was used for all experiments. Synthesized nanoparticles were characterized through Cryo-transmission electron microscopy (Cryo-TEM instrument FEI Tecnai T-20). UV-Vis spectroscopy was performed at a path length of 1 cm using Agilent Cary 60 UV-Vis spectrophotometer. Cary Eclipse Fluorescence Spectrophotometer (Agilent) with 5/10 slit width of excitation/emission was used to calculate the optical brightness of nanoparticles. Dynamic light scattering and zeta potential measurements were recorded by using (DLS)-BI200SM, Brookhaven Instruments Corporation, USA. Fluorescence microscopy was carried out by using blue and red filters from an Inverted Fluorescent Microscope. Digital pictures were captured by using an iPhone12 camera. Preparation of ultrabright biomimetic nanovesicles (BNVs). Cancer cell membrane nanovesicles based ultrabright particles were prepared from MCF-7 cancer cells (7×10 5 cancer cells/experiments). In brief, over confluent cancer cells were washed with PBS (pH 7.4) followed by centrifugation (10,000 for 20 minutes) and then further washed with ice-cold Tris-Magnesium buffer. The obtained pellet was homogenized for 10 minutes and then treated for 3 h followed by a Freeze-Thaw process. The freeze-thaw process was repeated 4–5 times and then obtained cells were centrifuged (10,000) for 60 minutes. The collected pellet was diluted with 20 mL of PBS and 10 mL of Tris-Magnesium buffer and washed thoroughly by centrifugation. The obtained colorless pellet was treated with 1X PBS and 50 mM Tris-Magnesium buffer for 6 h and then 0.1X PBS for overnight. Further, the collected pellet from the suspension, as mentioned above was treated with hypotonic (a mixture of 50 mM Tris-Magnesium buffer, 0.2% HCL, and 0.1X PBS) suspension for 12 h. The supernatant containing small bilayers of ruptured ghosts’ cells was collected by centrifugation (10,000 rpm for 60 minutes) and diluted with 10 mL PBS (pH 7.4). Now, the diluted suspension was allowed to self-assembled which was further treated under probe sonication process (20 cycles) with 40% intensity and 10 seconds on/off the pulse and filtered under high pressure to achieve the nanosized cell vesicles. For engineering ultrabright nanovesicles, red emissive amphiphilic miniature J-aggregates of dye molecules were prepared from 4 weeks old stock solution of dye (0.02 mM in the aqueous medium) which was treated at high temperature (60°C) for 2 h. These J-aggregates of dye were introduced in the mixture of small bilayers of ruptured ghosts’ cells and then allowed them for self-assembly before the sonication process (20 cycles) with 40% intensity and 10 seconds on/off the pulse). J aggregated ICG tarped biomimetic nanovesicles were dialyzed for 2 days against DI water (with dialysate changing every 12 h) and characterized further. The same process was also used to encapsulate the monomers of ICG (freshly prepared 0.01 mM in the aqueous medium at room temperature) in biomimetic nanovesicles. Amphiphilic cargo encapsulation within biomimetic nanovesicles. To understand the characteristics of lipid bilayer in cancer cell derived nanovesicles, J aggregates of lipophilic and red emissive Nile Red dye (0.01 mM in DMSO) were introduced in the mixture of small bilayers of ruptured ghosts’ cells and then allowed them for self-assembly for overnight. This suspension was treated for the sonication process (20 cycles) with 40% intensity and 10 seconds on/off the pulse). To examine, the presence of surface biomarkers/proteins on the exterior surface of biomimetic nanovesicles, ICG dye (100 µL) was incubated with 500 µL of membrane nanovesicles (5 mg/mL) for overnight at ambient temperature followed by slow mixing. The obtained mixture was centrifuged (6000 rpm) for 5 minutes and dialyzed for 2 days (12 kD membrane) against DI water in the dark conditions. Aqueous dispersibility, phototransduction, photosensitizing ability and photostability. Various formulations of dye and drug tagged nanovesicles (100 µL) were mixed in an aqueous suspension (1 mL of PBS). These samples were tested for dispersibility at different time points (1st day to 30th day) followed digital photography measurements at room temperature conditions which were corroborated by spectroscopic measurements. For photostability measurements, the dye integrated biomimetic nanovesicles (0.2 mg/mL) were tested for spectroscopic study by measuring their emission properties at various time points (1 h to 30 days) with and without treating them in serum. Single membrane ghost nanovesicles accommodate a few hundred dye molecules which help in demonstrating better and controlled emission properties whereas free dye molecules lose their optical behavior due to light sensitivity. Fluorescence intensity and stability of dye tagged nanovesicles were recorded under 760 nm excitation. Time dependent (0–5 minutes) phototransduction performance of designed optotheranostics (150 µL volume) was measured in an individual well of 96-well plate followed by NIR light irradiation (808 nm, 1 W power). Prior to phototransduction experiments, the water bath temperature was stabilized at 37.0°C. The temperature response was recorded in each 1 minute. For photosensitizing response of dye tagged nanovesicles, various concentrations (10–200 µg/mL) of these vesicles were treated with NIR light. After complete NIR irradiation, these vesicles are stained with DCFDA (2′,7′-dichlorofluorescin diacetate) to measure the produced ROS. In vitro biocompatibility and cancer cell imaging. L929 (5×10 5 cells per treatment) cell viability test was demonstrated at various concentrations (10–500 µg/mL) of nanovesicles (BNVs), ICG encapsulated BNVs, Nile Red encapsulated BNVs, free ICG and Nile Red followed by MTT assay. In brief, normal cells were seeded and incubated for 24 h with 5% CO 2 in Dulbecco’s Modified Eagle’s Medium (DMEM Gibco, Carlsbad, CA, USA) supplemented with 10% Fetal Bovine Serum and penicillin/streptomycin at 37.0°C. After 24 h incubation, these cells were treated for further 18 h with 40 µL of each nanoparticle (10–500 µg/mL) and then washed with PBS to remove excess particles from the treatment. To examine % cell viability, 20 µL of MTT dye was added to the above treatments and protected from light. Now, formazan crystals which were formed during the treatments were dissolved using 200 µL of DMSO. The optical absorbance was recorded using microplate reader (Tecan Infinite 200 PRO). Untreated cells were considered as control for calculating the % cell viability from the MTT assay. For cancer cell imaging and uptake studies, three different breast cancer cells (MCF-7, MDA-MB231 and 4T1, 5×10 5 cells/well) were cultured in DMEM culture media supplemented with 10% Fetal Bovine Serum and penicillin/streptomycin followed by 24 h incubation in 5% CO 2 at 37.0°C. After the complete culture of cancer cells, 40 µL of dye (ICG and Nile Red) encapsulated nanovesicles were treated with breast cancer cells for 3 h. These treated cancer cells were washed with PBS several times to remove the unattached or free nanoparticles. Now, dye tagged nanovesicles (BNVs) treated cells were fixed with 4% paraformaldehyde and stained with 4, 6-diamidino-2-phenylindole (DAPI for cell nuclei). These treated and stained cells were mounted with the cover slip using 70% glycerol on a glass slide and then brought under fluorescence microscope to track the location of red dots (from dye tagged nanovesicles) and blue nuclei. Solid tumor imaging and in vivo bio-distribution. In vivo protocol in 6 weeks old female Balb/c mice (~ 20 g weight) was approved by the Institutional Animal Ethical Committee. All experimental protocols on Balb/c female mice were approved by the Institutional Animal Ethical Committee (IAEC) of the National Centre for Cell Science, Pune, India (NCCS, Pune). Imaging studies were performed during anesthetized condition (50 µL dose with 1:4 of Xylazine and Ketamine for anesthesia). For solid tumor development, 5 x 10 5 4T1 cells were injected subcutaneously into the mammary fat pad of female mice and kept for about 15 days of tumor growth observation which was measured using a caliper. On the 15th day of tumor growth, a single dose (10 mg/kg body weight containing 2.5×10 13 nanoparticles/cm 3 ) of three different nanoparticles (i. ICG J-aggregates biomimetic nanovesicles (interior), ii. ICG J-aggregates biomimetic nanovesicles (exterior) and iii. Nile Red dyes J-aggregates within lipid layers of biomimetic nanovesicles) were intravenously injected in mice. Nanoparticles administrated (10 mg/kg dose) tumor bearing animals were placed for whole body Near Infrared Fluorescence (NIRF) Imaging and scans were demonstrated for different time points (0.5 h, 1 h, 3 h, 6 h, 12 h and 24 h) under anesthetized conditions (50 µL dose with 1:4 of Xylazine and Ketamine for anesthesia). Fluorescence signals coming from nanoparticles were measured from whole body including tumor area and vital organs (heart, liver, spleen, and kidneys). Biodistribution measurements were evaluated by considering quantitative ROI values of the fluorescence intensity of the corresponding organs and tumors in vivo . All imaging scans were done in anesthetized condition (50 µL dose of Xylazine and Ketamine for anesthesia) of animals (n = 3). Several qualitative and quantitative analyses were done and compared with pre-injected animals (control) to evaluate the site-selective tumor imaging and biodistribution. Animals were euthanized under 5 minutes of CO 2 inhalation to collect tissues and tumor samples for ex-vivo and pathological examinations at different time points. Additionally, some animals were sacrificed after 24 h of injection and major organs were collected and tested for tissue pathological examinations (hematoxylin and eosin, H&E) which were conducted for in vivo toxicity and to investigate the tissue injuries of injected nanoparticles. In vivo toxicity and clearance measurements. All treated mice were sacrificed after injecting nanoparticles and collected major organs and tumors were made for histopathological examinations (Hematoxylin and Eosin, H&E) to study the tissue damage. Additionally, body weight measurements, health behavior, hair loss, eschars and inflammation on animal’s body of treated animals were also examined to ensure the adverse effect of injected nanoparticles. Dye per nanoparticle and effective brightness calculation Before calculating the nanoparticles per cm 3 , dye per nanoparticles and their relative brightness, concentration of engineered dye tagged nanovesicles was measured after drying (48–50°C) a known volume (150 µL) of nanoparticles (60 nm in diameter) followed by dry residue of used solvent as a baseline. The mass of the particle per unit volume of the solution was 0.3 mg/mL with 1.36 x 10 − 13 mg weight of one nanoparticle. Further, we have calculated the number of particles (7.3 x 10 11 nanoparticles/cm 3 ) in the stock solution using the mass density (1210 mg/cm 3 ) and diameter of nanovesicles (60 nm). Spectroscopic methods were applied to estimate the number of dye molecules per cm 3 (5.74 x 10 13 dye molecules/ cm 3 ) and dye per nanoparticles (786) considering extinction coefficients 2.621 x 10 5 M − 1 cm − 1 at 780 nm and Beer–Lambert law. Importantly, the same concentration of free dye was considered and absorption of free nanovesicles was subtracted for estimating these numbers including relative brightness (737). We have measured the fluorescence which was coming from the solution of ICG encapsulated nanovesicles (known concentration) and the reference dyes ICG. To estimate the numbers of cargo molecules loaded within a single nanovesicle viz., BNV, we need to estimate the mass of a single nanovesicle considering radius (r) and using the equations: Statistics . For statistical analysis, all experiments were performed in triplicate. All data were analyzed and plotted using OriginPro 8. Significant observations between different groups were calculated by t-test. ASSOCIATED CONTENT Supporting Information. cryo-TEM images, DLS measurement, UV-Vis and emission spectra, equations for drug loading efficiency, ultra brightness and photostability data, Zeta potential measurement, digital photographs, release kinetics data, and experimental setup, cell imaging, in vivo distribution and whole-body scans. Abbreviations NIR, Near-Infrared, density, concentration, DLS, dynamic light scattering, λ abs/em represent absorption/emission, A absorbance, ε extinction coefficient, TEM, transmission electron microscopy, FLBNV fluorescence from BNV, CBNV concentration of BNVs, FL Dye fluorescence from organic dyes, C Dye concentration of organic dyes, r radius of single BNV, MESF units (Molecules of Equivalent Soluble Fluorochrome), ICG Indocyanine green was named as ICG. Declarations Author Contributions R.P. has designed the project and has done major experiments for engineering nanoparticles, and their spectroscopic characterization. K.P. contributed to providing cells, washing them, and cell imaging studies. M.T. has done some preparation for nanoparticles and has performed cryo-TEM of prepared nanovesicles. J.B. has helped for cryo-TEM imaging. M.G. P.B. and G.C.K. have done in vitro and in vivo studies. V.K.D has provided the cells and related facility. P.C. and R.K. have helped in the manuscript preparation. S.K. has helped in in-vivo analysis. All authors have contributed to writing and revising the draft and have approved the final version of the manuscript. $ These authors have contributed equally to this project. Competing interests R.P. holds patents related to lipid, gold and silica nanoparticles. All other authors declare no competing financial interest. ACKNOWLEDGMENT R.P. thanks the director and the School of Biochemical Engineering IIT (BHU) Varanasi for providing the necessary facilities and support. This work was supported by the Departmental Instrumentation and Laboratory facilities. The authors would like to thank the Central Discovery Centre (CDC) and SATHI, IIT (BHU) for cell-imaging studies. We thank Sahu Bio Tech Services, Kirkatwadi, Pune, Maharashtra, India, for laser support and suggestions during the project. 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visible and NIR (C) and chemical formulations of ICG monomer and their aggregates (D).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4616433/v1/a768027cb56190a57566bf25.png"},{"id":62137519,"identity":"ad307115-f3fd-4a63-83b1-45097f7583b1","added_by":"auto","created_at":"2024-08-09 16:34:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":452469,"visible":true,"origin":"","legend":"\u003cp\u003eCartoons and respective microscopic images (Cryo-TEM) of red emissive ICG and Nile Red dyes J-aggregates within biomimetic nanovesicles designed from breast cancer cells. J-aggregates of ICG dye are located in interior and exterior surface of nanovesicles and J-aggregates of Nile Red dye molecules are embedded within the bilayer of ghost nanovesicles (A). UV-Vis-NIR absorption spectra of amphiphilic and NIR emissive J-aggregates of ICG dye located in interior and exterior surface of nanovesicles and comparison with monomers of used organic dye molecules (B). J-aggregates of ICG dye within nanovesicles are showing bathochromic shift (893-896 nm) from their original absorption peak (780 nm). NIR emission spectra of amphiphilic J-aggregates of ICG tagged nanovesicles and comparison with monomers of used organic dye molecules, empty nanovesicles at different time points (C). pH dependent % release kinetics of ICG dye molecules from engineered nanovesicles showing their cargo delivery efficacy in cancer mimicked environment (D) and J-aggregates of ICG dye (located in interior and exterior surface of nanovesicles) and J-aggregates of Nile Red dye molecules (embedded within the bilayer of ghost nanovesicles) for breast MDA-MB231 cancer cell imaging (E).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4616433/v1/66e004edd613754840d58bfd.png"},{"id":62138191,"identity":"594fed80-3142-4227-88a0-4a53500ebe77","added_by":"auto","created_at":"2024-08-09 16:42:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":243873,"visible":true,"origin":"","legend":"\u003cp\u003eJ-aggregates of ICG dye (located in interior and exterior surface of nanovesicles) and J-aggregates of Nile Red dye molecules (embedded within the bilayer of ghost nanovesicles) for single cell imaging of 4T1 breast cancer cell (A), Brightness measurements of J-aggregates of ICG dye-nanovesicles (B) and % Cell cytotoxicity measurements of engineered dye tagged nanovesicles and their components viz., empty nanovesicles (BNVs), ICG monomer encapsulated BNVs, ICG J aggregate encapsulated BNVs, free ICG, free Nile Red (C) with normal L929 cells.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4616433/v1/3bf9d1eb3e68e34fc3bff83d.png"},{"id":62137520,"identity":"d7ba57b1-ea73-48f6-96ad-6f21199b4bba","added_by":"auto","created_at":"2024-08-09 16:34:02","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":650972,"visible":true,"origin":"","legend":"\u003cp\u003eTime dependent (0.5 h to 24 h) site-selective solid tumor and metastasis imaging in tumor bearing mice after systemic administration of J-aggregates of ICG dye (located in interior and exterior surface of nanovesicles, A, B) and biodistribution measurements after 24 h of post-administration (C) and histopathology examination for tissue toxicity with the vital organ’s appearance (D).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4616433/v1/1158c1cab235497c2951682d.png"},{"id":62138897,"identity":"62445f3d-5080-4c55-acd9-816025dc0c8e","added_by":"auto","created_at":"2024-08-09 16:50:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2306612,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4616433/v1/7f0fc682-3f44-436b-8c5c-71e91f5a62ff.pdf"},{"id":62137522,"identity":"4a2bf64d-60ce-4ee1-8b4c-3bef76b11190","added_by":"auto","created_at":"2024-08-09 16:34:04","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":59341467,"visible":true,"origin":"","legend":"","description":"","filename":"ESIUltrabrightNVsIITBHU.docx","url":"https://assets-eu.researchsquare.com/files/rs-4616433/v1/ceb11dae215e1d8dc7aea077.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"NIR Emissive Biomimetic Ghost Nanovesicles for Site-Selective Solid Tumor Imaging","fulltext":[{"header":"Introduction","content":"\u003cp\u003e\u003cstrong\u003eN\u003c/strong\u003eear-infrared responsive fluorescence (NIRF) imaging has been considered as versatile and safe approach for visualizing abnormal tissues/tumor selectively in pre-clinical and clinical models.\u003csup\u003e1–3\u003c/sup\u003e Among non-invasive contrast agents, optically active imaging probes offer several advantages for site-selective diagnosis of solid tumors, assessing the blood perfusion, real-time cancer lymphography, etc., due to deep tissue penetration, low light scattering, and high spatial resolution of the near-infrared light.\u003csup\u003e4–8\u003c/sup\u003e Moreover, NIR emissive nanoprobes exhibit low photon absorption and light scattering, resulting in a high signal-to-noise ratio with promising diagnostic ability.\u003csup\u003e9–14\u003c/sup\u003e Indocyanine green (ICG, NIR fluorescent dye)\u003csup\u003e9\u003c/sup\u003e is one such imaging probe approved by the U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA) for pre-clinical and clinical applications.\u003csup\u003e15\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e16\u003c/sup\u003e Overall, ICG dye has been recognized as a promising contrast agent with low interference from tissue autofluorescence and blood absorption (500-600 nm) due to absorption and emission in the NIR spectral range (650-900 nm).\u003csup\u003e17\u003c/sup\u003e Apart from cancer diagnosis, ICG dye has been applied to examine the various cardiac, liver, and ophthalmic angiography functions due to enhanced fluorescence intensity of plasma-integrated ICG aggregates\u003csup\u003e18–21\u003c/sup\u003e To the best of our knowledge and based on the literature survey, we can note that the fluorescence intensity and lifetime may fluctuate with varying plasma concentrations (protein and lipoprotein).\u003csup\u003e14,15\u003c/sup\u003e Nonspecific biodistribution, low blood circulation time, and photodegradation of free organic ICG dye are major limitations in medical applications.\u003csup\u003e22\u003c/sup\u003e These amphiphilic ICG molecules show molecular aggregation in the aqueous environments, resulting in fluorescence quenching\u003csup\u003e23\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e24\u003c/sup\u003e,which diminishes/reduces their fluorescence and light mediated therapeutics ability. Recently, aggregation-induced emission (AIE)\u003csup\u003e25\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e26\u003c/sup\u003e has been considered a new development in fluorophores, which resolves the problem of aggregation-induced quenching of organic imaging probes. Particularly, J-aggregates of organic dyes (slip-stacked alignment)\u003csup\u003e27\u003c/sup\u003e have been used for bioimaging due to delocalized π-electrons, which leads to constructive coupling of the excited state transition dipoles (Figure 1A-D). In spectroscopic manner, organic dye aggregates (J-aggregates) produce bathochromic shift in absorption and emission with narrow bands, enhanced molar extinction coefficient (ε), and shortened fluorescence lifetimes with better quantum yields (ΦF) which decide their average brightness.\u003csup\u003e28–31\u003c/sup\u003e Aggregates of organic dyes like squaraine, flavylium, porphyrin, perylene bisimide derivatives, heptamethine cyanine, and pyrrolopyrrole cyanine have been reported but limited with low photostability, easy degradation, nonspecific biodistribution and binding which limit their ability for site-selective tumor imaging.\u003csup\u003e31–36\u003c/sup\u003e Therefore, encapsulating these emissive molecules in the nanoparticles can resolve the aforementioned obstacles. For example, in 2016, aggregate porphyrin-lipids nanoparticles were tested for image-guided surgery.\u003csup\u003e37\u003c/sup\u003e Recently, squaraine J-aggregate-polymeric micelles have been tested for image-guided photothermal therapy.\u003csup\u003e35\u003c/sup\u003e However, premature leakage of loaded emissive molecules, poor brightness, low colloidal stability, and lack of specific, and natural tumor targeting ability are remaining challenges of these nanosized formulations.\u003csup\u003e38–40\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eHerein, we report cancer cell derived biomimetic ghost nanovesicles entrapping amphiphilic dye aggregates (ICG and Nile Red) within bilayers, internal cavity, and exterior surface, demonstrating their structural characteristics. Further, these engineered optically active biomimetic ghost nanovesicles were tested for solid tumor imaging in pre-clinicalmodels. For these optically active biomimetic imaging agents, aqueous solution of dye (0.01 mM in the aqueous medium) has been used to prepare fluorescent amphiphilic dye aggregates whereas monomers of dye have been tested as photosensitizer.\u003c/p\u003e"},{"header":"RESULTS AND DISCUSSION","content":"\u003cp\u003eCharacteristics of dye tagged biomimetic nano ghost vesicles are evaluated through microscopic and spectroscopic measurements which are further corroborated by \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e studies (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-E). Dye integrated (J-aggregates) ghost nanovesicles (~\u0026thinsp;60 nm) show NIR (650\u0026ndash;900 nm) absorption and emission which have been tested for solid tumor imaging and localized biodistribution. Engineered optically active biomimetic imaging agents\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e are stable for several days (tested up to 30 days, see supporting information for more details) showing promising brightness (779 MESF for 784 ICG/BNV and 2.69 \u0026times; 10\u003csup\u003e11\u003c/sup\u003e BNVs/cm\u003csup\u003e3\u003c/sup\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and Table\u0026nbsp;1) for localized tumor visualization. Moreover, phototransduction response of designed biomimetic optically active ghost nanovesicles (J-aggregates and monomers of ICG dye entrapped nanovesicles) have been evaluated due to their NIR absorption ability. Upon 5 minutes of NIR light exposure, J-aggregates of ICG dye demonstrate better phototransduction (57\u0026deg;C at 2.5\u0026times;10\u003csup\u003e13\u003c/sup\u003e nanoparticles/cm\u003csup\u003e3\u003c/sup\u003e) compared to ICG monomers (44\u0026deg;C at 2.5\u0026times;10\u003csup\u003e13\u003c/sup\u003e nanoparticles/cm\u003csup\u003e3\u003c/sup\u003e). Whereas ICG monomers tagged biomimetic nanovesicles exhibit better photosensitizing property as compared to J-aggregates encapsulated nanovesicles (details are discussed below). On the other hand, we have noticed that ICG J-aggregates encapsulated nanovesicles exhibit better brightness (754 MESF units) than their monomer (598 MESF units) may be due to self-quenching of free monomers in nanovesicles. Additionally, it is believed that monomers of small organic dye molecules can diffuse easily from the interior of nanovesicles as compared to their aggregated form. Hence, they may exhibit poor emission property which may not be suitable for bioimaging applications. It should be noted that nanovesicles have lipid assemblies in their membrane. Hence, lipid specific dye remains in the membrane for a long time (at least 12\u0026ndash;24 h) due to lipophilic nature of small organic molecules. In the present work, we have used Nile Red, which is specific to the lipid layers with BNVs, but they require high numbers of dye molecules per particle (802) and a number of BNVs per cm\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e (7.90 \u0026times; 10\u003csup\u003e12\u003c/sup\u003e) to exhibit comparable brightness (748 MESF units) to the ICG J-aggregates in BNVs due to molar extinction coefficient (ε, 38000 cm\u003csup\u003e-\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e M\u003csup\u003e-\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e) barriers. Cancer cell membrane ghost vesicles\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e (3\u0026ndash;5 mg/mL) are prepared from breast cancer cells (5\u0026times;10\u003csup\u003e5\u003c/sup\u003e-7\u0026times;10\u003csup\u003e5\u003c/sup\u003e cancer cells/experiments) in hypotonic conditions treated with various buffers (ice-cold Tris-Magnesium and PBS) followed by freeze-thaw and centrifugal process (Supplementary Fig.\u0026nbsp;1A). These ghost vesicles are used to prepare nanosized biomimetic cell membrane vesicles under multiple rounds of probe sonication (20 cycles) in ice bath. Uniform size distribution (~\u0026thinsp;60 nm, Supplementary Fig.\u0026nbsp;1B) of prepared biomimetic cell membrane vesicles has been examined by cryo-mode TEM, which is corroborated by dynamic light scattering measurements (0.28 PDI) (Supplementary Fig.\u0026nbsp;1C). Diameter sharpness in light scattering measurements indicates nanovesicles' spherical morphology, which is clearly ensured by microscopic imaging. On the other hand, the negative surface charge (-12 mV) of nanovesicles supports their colloidal stability in physiological conditions, which may be due to the presence of natural surface biomarkers (Supplementary Fig.\u0026nbsp;1D). These available inherent surface biomarkers, mostly proteins, help in (i) better particle dispersion in the physiological environment observed from microscopic images shown in Supplementary Fig.\u0026nbsp;1E, (ii) natural targeting, and (iii) evading macrophage uptake (data are not shown here). Interestingly, controlled particle diameter (~\u0026thinsp;68 nm) in the aqueous medium indicated the better dispersion of nanovesicles over 30 days of storage (Supplementary Fig.\u0026nbsp;1E and Supplementary Fig.\u0026nbsp;2A).\u003c/p\u003e \u003cp\u003eMore importantly, we are trying to address major hurdles such as (i) are these cells derived nanovesicles exhibit homogeneous size distribution and (ii) what is the fate of repeatability and scalability of these biomimetic nanovesicles? Secondly, we also have tried to understand the numbers of encapsulated dye molecules which decides the brightness of particles. To address these critical challenges, we have optimized the preparation recipe and made sure the reproducibility for more than 10 times. However, we have noticed slight changes in the final product concentration (1.20 to 1.32 mg/mL), particles size (60 to 65 nm) distribution and numbers of dye molecules per nanovesicle (201 to 281) during each batch (see Supplementary Table\u0026nbsp;1 and Table\u0026nbsp;2 in the supporting information). One the other hand, batch scalability is also a major challenge especially for liposomes and cell derived biomimetic nanovesicles which has been specifically taken care while optimizing the present recipe. We have noticed that concentration of magnesium chloride and sucrose in Tris buffer and Freeze-Thaw time are essential components apart from mechanical forces which decide the cell ghost structures and surface biomarkers. Results of 10 different batches demonstrate the consistency in final product concentration (1.20 to 1.32 mg/mL concentration per 15 mL batch). It should be noted that the biomimetic ghost nanovesicles have been achieved with 5 mg/mL maximum yield but that was for large scale production (100 mL volume of cell suspension) where 65 nm size of nanovesicle can encapsulate 215 ICG dye molecules in its cavity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv id=\"Equa\" class=\"Equation\"\u003e \u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$Weightof 1 BNV \\left(mg\\right)=\\rho \\times 4/3\\pi r^3 \\left(1\\right)$$\u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Equb\" class=\"Equation\"\u003e \u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\n$$NumberofBNVs/mL=C/(\\rho \\times 4/3\\pi r^3) \\left(2\\right)$$\u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Equc\" class=\"Equation\"\u003e \u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equc\" name=\"EquationSource\"\u003e\n$$NumberofDyes/mL=A/ \\epsilon \\times 6.023\\times 10^23 \\left(3\\right)$$\u003c/div\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003eSo far, several studies have been reported on cell membrane-derived biomimetic nanovesicles and their integration with fluorescent dye molecules.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e However, ultra brightness and the importance of integrated dye for the structural characteristic of membrane nanovesicles have yet to be achieved. In the current study, we show the amphiphilic dye molecules (ICG and Nile Red) integrated biomimetic ghost nanovesicles at the nanoscale for targeted tumor imaging and specific biodistribution. In microscopic\u003c/p\u003e \u003cp\u003eimages, observed dark patches from the (i) cavity (505 to 784 ICG molecules/nanovesicle), (ii) exterior surface (578 to 828 ICG molecules/nanovesicle), and (iii) bilayer of single nanovesicle represent the assembly of amphiphilic dye molecules (ICG and Nile Red) in the form of J-aggregates (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Details of nanovesicle numbers, dye per nanovesicles and respective brightness are thoroughly discussed in the supporting information (Supplementary Table\u0026nbsp;1). These observations are further corroborated with spectroscopic measurements, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, C. In absorption spectra, aqueous ICG monomers (0.01 mM) exhibit λ\u003csub\u003emax\u003c/sub\u003e absorbance at 780 nm and a weaker H-aggregate peak at 710 nm. Aqueous dye at high temperature (60\u0026deg;C) incubation result in miniature J-aggregates showing bathochromic shifts in absorption and emission (λ\u003csub\u003eabs/emm\u003c/sub\u003e 890/804 nm) with peak sharpness.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurther, these J-aggregates exhibit significant bathochromic shifts in absorption and emission (λ\u003csub\u003eabs/emm\u003c/sub\u003e 893/806 nm) upon their integration with biomimetic cell ghost nanovesicles which are purified through dialysis (for 2 days using 12 KD dialysis membrane). Photo stability of dye tagged nanovesicles have been evaluated by measuring their emission properties at various time points (1 h to 30 days) with and without treating in serum (Supplementary Fig.\u0026nbsp;2B, C). It has been calculated that a single membrane ghost nanovesicles accommodate a few hundred dye molecules exhibiting ultra brightness (498 to 778 MESF units viz., Molecules of Equivalent\u003c/p\u003e \u003cp\u003eSoluble Fluorophore). Controlled emission and maintained brightness for a prolonged period (tested up to 30 days) demonstrate the photostability of emissive ghost nanovesicles, whereas free dye J-aggregates demonstrate\u0026thinsp;~\u0026thinsp;40% degradation and reduced emission (Supplementary Fig.\u0026nbsp;2A-C). Overall, the formed J-aggregates demonstrate better fluorescence intensity and stability under 760 nm excitation when they are within the nanoparticulate formulation.\u003c/p\u003e \u003cp\u003eIt should be noted that free ICG in aqueous solution mainly exhibit their monomers with two absorption peaks at 780 and 712 nm, respectively whereas these monomers turn into aggregated form (J aggregates, a sharp extinction peak at 890 nm) due to noncovalent π\u0026ndash;π stacking and hydrophobic interactions upon 60\u0026deg;C incubation for 2 h. Further, these ICG J-aggregates integrated ghost nanovesicles exhibit hyperthermia temperature (43\u0026deg;C at 2.5\u0026times;10\u003csup\u003e13\u003c/sup\u003e nanoparticles/cm\u003csup\u003e3\u003c/sup\u003e) within 2 minutes which is further improved to 57\u0026deg;C after 5 minutes of NIR exposure compared to ICG monomers encapsulated nanovesicles (44.6\u0026deg;C at 2.5\u0026times;10\u003csup\u003e13\u003c/sup\u003e nanoparticles/cm\u003csup\u003e3\u003c/sup\u003e) as shown in Supplementary Fig.\u0026nbsp;2D. Whereas ICG monomers tagged biomimetic ghost nanovesicles exhibit better photosensitizing property (80\u0026ndash;85%) as compared to J-aggregates encapsulated nanovesicles (20%, see Supplementary Fig.\u0026nbsp;2E) which indicate their photodynamic therapeutics applicability (data not shown here).\u003c/p\u003e \u003cp\u003eNext, microscopic characteristics reveal that amphiphilic ICG J-aggregates integrated nanovesicles exhibit the exterior dark layer (5\u0026ndash;6 nm) around due to the presence of aggregated dye molecules within the available surface biomarkers of nanovesicles which is corroborated by spectroscopic measurements (λ\u003csub\u003emax\u003c/sub\u003e, absorbance at 892\u0026ndash;893 nm and λ\u003csub\u003emax\u003c/sub\u003e, emission at 806 nm) as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, C. Similarly, the presence of lipid bilayers (2\u0026ndash;3 nm) within biomimetic nanovesicles is physiochemically characterized. J-aggregates of lipophilic Nile Red dye in nanovesicles show bathochromic shifts (34 nm red shift) with λ\u003csub\u003emax\u003c/sub\u003e, absorbance at 604 nm, and emission at 662 nm as compared to their monomers (λ\u003csub\u003emax\u003c/sub\u003e, absorbance at 570 nm, Supplementary Fig.\u0026nbsp;3) which is also supported by microscopic characterization. The presence of amphiphilic J-aggregates in nanovesicles is validated through release kinetic patterns in cancer mimicked conditions viz., acidic conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Dye from the exterior surface of vesicles show rapid release as compared to J-aggregates inside the vesicles (Supplementary Fig.\u0026nbsp;4 and Supplementary Fig.\u0026nbsp;5). These nanovesicles demonstrate better cargo release (more than 10%) in late endosomal (pH 2\u0026ndash;4) and tumor mimicked environment (pH 6.5) as compared to physiological environment (less than 10% at pH 7.4). Further, the red patches from cancer cells (MDA-MB231 and 4T1) interior indicate the presence of red emissive dye aggregates (ICG and Nile Red) within the BNVs located at different nanovesicle sites. Inherent surface biomarkers of nanovesicles exhibit strong binding with breast cancer cells (MDA-MB231), indicating their natural targeting ability shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, and Supplementary Fig.\u0026nbsp;6. Empty ghost nanovesicles show distinct cavities as compared to J-aggregates integrated nanovesicles confirmed through microscopic characterization (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, Supplementary Fig.\u0026nbsp;1B, and Supplementary Fig.\u0026nbsp;7). These characteristics are discussed here for the first time in the case of cell membrane-derived biomimetic imaging agents.\u003cdiv id=\"Equd\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equd\" name=\"EquationSource\"\u003e\n$$\\text{R}\\text{e}\\text{l}\\text{a}\\text{t}\\text{i}\\text{v}\\text{e} \\text{b}\\text{r}\\text{i}\\text{g}\\text{h}\\text{t}\\text{n}\\text{e}\\text{s}\\text{s}=\\frac{\\frac{\\text{F}\\text{L}\\text{B}\\text{N}\\text{V}}{C\\text{B}\\text{N}\\text{V}}}{\\frac{\\text{F}\\text{L}\\text{D}\\text{y}\\text{e}}{C\\text{D}\\text{y}\\text{e}}} \\left(4\\right)$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eFurther, these bright nanovesicles have also been tested for targeted cancer cell imaging (4T1 metastatic breast cancer cells), as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA. After 3 h of post-treatment, red emissive dye-tagged nanovesicles are accumulated in cancer cells. It is observed that the brightness of dye-tagged nanovesicles is dependent on the number of dyes per nanovesicle, which define as a multiplication of the quantum yields (ΦF) and the extinction coefficient ε(λ) (M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e units) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and equations 1\u0026ndash;4). In the present work, the brightness of nanoparticles is calculated by considering the 2.621\u0026times;10\u003csup\u003e5\u003c/sup\u003e M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e extinction coefficient at various concentrations of nanoparticles (0.9 to 2.5 mg/mL, Supplementary Table\u0026nbsp;1). The prepared ultrabright nanovesicles are tested for \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e biocompatibility. \u003cem\u003eIn vitro\u003c/em\u003e compatibility, BNVs before and after different cargo (J-aggregates of ICG and Nile Red) loading show more than 95% cell viability (200 \u0026micro;g/mL) of healthy cells which is recorded 80\u0026ndash;85% at 500 \u0026micro;g/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). It should be noted that engineered BNVs demonstrate multifunctional abilities due to entrapped different cargo molecules (ICG, and Nile Red), which are examined through physicochemical studies and release kinetics (negligible release is seen at neutral pH 7.4), indicating safe entrapment of loaded cargo molecules (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Supplementary Fig.\u0026nbsp;8 and Supplementary Fig.\u0026nbsp;9). Brightness and contrast response of J-aggregates (ICG and Nile Red) tagged BNVs have been evaluated in tumor-bearing female mice at different concentrations (0.1-1 mg/mL).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eRemarkably, ICG J-aggregates-tagged BNVs demonstrate 20-fold brightness compared to only ICG J-aggregates in the aqueous medium as shown in Supplementary Fig.\u0026nbsp;10. To evaluate the site-selective tumor imaging and specific biodistribution, a single dose (2.5\u0026times;10\u003csup\u003e13\u003c/sup\u003e nanoparticles/cm\u003csup\u003e3\u003c/sup\u003e) of J-aggregates (ICG and Nile Red) tagged BNVs are intravenously administrated to 4T1 tumor-bearing female Balb/c mice. After post-injection, whole-body scans are captured through \u003cem\u003ein vivo\u003c/em\u003e imaging system at different time points (0.5 h to 24 h), indicating a clear visualization of the tumor site with better brightness signal intensity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-C). In initial post-injection time (0.5 h to 1 h) the injected ultrabright BNVs contrast agents adjust their distribution (0.98\u0026times;10\u003csup\u003e8\u003c/sup\u003e to 0.23\u0026times;10\u003csup\u003e8\u003c/sup\u003e p/sec/cm\u003csup\u003e2\u003c/sup\u003e/sr) among vital organs (heart, liver, spleen) and increase in the tumor\u003c/p\u003e \u003cp\u003esite (0.065\u0026times;10\u003csup\u003e8\u003c/sup\u003e to 1.98\u0026times;10\u003csup\u003e8\u003c/sup\u003e p/sec/cm\u003csup\u003e2\u003c/sup\u003e/sr) from 3 h of post-injection time which is further constant (2.2\u0026times;10\u003csup\u003e8\u003c/sup\u003e to 2.6\u0026times;10\u003csup\u003e8\u003c/sup\u003e p/sec/cm\u003csup\u003e2\u003c/sup\u003e/sr) up to 24 h of post-injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-C and Supplementary Fig.\u0026nbsp;11 and Supplementary Fig.\u0026nbsp;12). On the other hand, it has been noted that ICG J-aggregates-BNVs (exterior) exhibit higher signal intensity and brightness (2.4\u0026times;10\u003csup\u003e8\u003c/sup\u003e p/sec/cm\u003csup\u003e2\u003c/sup\u003e/sr) from the tumor site as compared to ICG J-aggregates-BNVs (interior) (2.0\u0026times;10\u003csup\u003e8\u003c/sup\u003e p/sec/cm\u003csup\u003e2\u003c/sup\u003e/sr) indicating the quick release response of emissive dye from exterior surface of BNVs in the cancer mimicked environment as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA. Interestingly, we have noticed that injected ultrabright BNVs contrast agents identify the\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003emetastasis in tumor bearing mice. Moreover, Nile Red J-aggregates-BNVs also demonstrate promising signal intensity (1.4\u0026times;10\u003csup\u003e8\u003c/sup\u003e p/sec/cm\u003csup\u003e2\u003c/sup\u003e/sr) from the tumor site which augment (1.6\u0026times;10\u003csup\u003e8\u003c/sup\u003e to 2.6\u0026times;10\u003csup\u003e8\u003c/sup\u003e p/sec/cm\u003csup\u003e2\u003c/sup\u003e/sr) with post-injection time (3 h to 24 h). Controlled and uniform fluorescence from the tumor area indicates the better accumulation and distribution of injected red emissive biomimetic nanovesicles than the pre-injected mice (Supplementary Fig.\u0026nbsp;12 and Supplementary Fig.\u0026nbsp;13). Biodistribution measurements have been evaluated by considering quantitative ROI values of the fluorescence intensity of the corresponding organs and tumors \u003cem\u003ein vivo\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, C). Further, the promising brightness and fluorescence from the whole body demonstrate the better circulation of nanovesicles, which are entering and stay within solid tumors and exiting via draining tumor lymphatic and then re-entering in the blood circulation as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. However, the clear mechanism is not known yet.\u003c/p\u003e \u003cp\u003eNext, these injected nanovesicles are site-specific for solid tumor without affecting healthy tissues (heart, lung, liver, spleen and kidneys) showing their better biocompatibility due to their natural surface biomarkers and biomimetic ability. The tissue toxicity has been evaluated through histopathology examinations as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD. Tissue histology exhibits no pathological damage or changes to the vital organs where (i) myofiber and muscle bundle in the heart, (ii) portal triad and central vein in the liver are noticed without any injury. Moreover, the glomerulus and tubules in the kidney are also without any histological change. On account of unique features, we can state that the engineered biomimetic ultrabright nanovesicles are safe to be used as contrast agents for localized tumor imaging and tissue visualization \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eIn summary, we present the safe bioengineering of cancer cell membrane derived biomimetic ghost nanovesicles which are tagged with amphiphilic dyes (ICG and Nile Red) aggregates for site-selective solid tumor imaging in pre-clinical models. Structural characteristics of these engineered biomimetic nanovesicles has been examined through microscopic and spectroscopic methods. These dye entrapped ghost nanovesicles exhibit intense brightness and signals intensity along with better phototransduction and photosensitizing ability. During diagnostics course, the primary tumor location and tumor metastasis have been clearly observed. Maintained body weight, long time survival and histopathology examinations ensure the \u003cem\u003ein vivo\u003c/em\u003e biocompatibility of these intravenously administrated biomimetic imaging agents. Among several reports, the structural features of engineered biomimetic nanovesicles have been discussed here, which are correlated with various physicochemical, \u003cem\u003ein vitro\u003c/em\u003e, and \u003cem\u003ein vivo\u003c/em\u003e studies. Hence, the developed cancer cell membrane vesicles could be considered clinically relevant imaging and therapeutics medicine.\u003c/p\u003e"},{"header":"EXPERIMENTAL SECTION","content":"\u003cp\u003e\u003cstrong\u003eMaterials and techniques\u003c/strong\u003e. Indocyanine green (ICG), Nile Red (NR), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) were purchased from Sigma-Aldrich. Dulbecco\u0026apos;s Modified Eagle Medium (DMEM), Phosphate buffered saline (PBS, pH 7.4), dimethyl sulphoxide (DMSO), Fetal bovine serum (FBS), 4\u0026rsquo;,6-diamidino-2-phenylindole (DAPI) and antibiotic-antimycotic solution were obtained from HiMedia Pvt. Ltd. Milli-Q (\u0026gt;\u0026thinsp;18.2 MΩ cm) was used for all experiments. Synthesized nanoparticles were characterized through Cryo-transmission electron microscopy (Cryo-TEM instrument FEI Tecnai T-20). UV-Vis spectroscopy was performed at a path length of 1 cm using Agilent Cary 60 UV-Vis spectrophotometer. Cary Eclipse Fluorescence Spectrophotometer (Agilent) with 5/10 slit width of excitation/emission was used to calculate the optical brightness of nanoparticles. Dynamic light scattering and zeta potential measurements were recorded by using (DLS)-BI200SM, Brookhaven Instruments Corporation, USA. Fluorescence microscopy was carried out by using blue and red filters from an Inverted Fluorescent Microscope. Digital pictures were captured by using an iPhone12 camera.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of ultrabright biomimetic nanovesicles (BNVs).\u003c/strong\u003e Cancer cell membrane nanovesicles based ultrabright particles were prepared from MCF-7 cancer cells (7\u0026times;10\u003csup\u003e5\u003c/sup\u003e cancer cells/experiments). In brief, over confluent cancer cells were washed with PBS (pH 7.4) followed by centrifugation (10,000 for 20 minutes) and then further washed with ice-cold Tris-Magnesium buffer. The obtained pellet was homogenized for 10 minutes and then treated for 3 h followed by a Freeze-Thaw process. The freeze-thaw process was repeated 4\u0026ndash;5 times and then obtained cells were centrifuged (10,000) for 60 minutes. The collected pellet was diluted with 20 mL of PBS and 10 mL of Tris-Magnesium buffer and washed thoroughly by centrifugation. The obtained colorless pellet was treated with 1X PBS and 50 mM Tris-Magnesium buffer for 6 h and then 0.1X PBS for overnight. Further, the collected pellet from the suspension, as mentioned above was treated with hypotonic (a mixture of 50 mM Tris-Magnesium buffer, 0.2% HCL, and 0.1X PBS) suspension for 12 h. The supernatant containing small bilayers of ruptured ghosts\u0026rsquo; cells was collected by centrifugation (10,000 rpm for 60 minutes) and diluted with 10 mL PBS (pH 7.4). Now, the diluted suspension was allowed to self-assembled which was further treated under probe sonication process (20 cycles) with 40% intensity and 10 seconds on/off the pulse and filtered under high pressure to achieve the nanosized cell vesicles. For engineering ultrabright nanovesicles, red emissive amphiphilic miniature J-aggregates of dye molecules were prepared from 4 weeks old stock solution of dye (0.02 mM in the aqueous medium) which was treated at high temperature (60\u0026deg;C) for 2 h. These J-aggregates of dye were introduced in the mixture of small bilayers of ruptured ghosts\u0026rsquo; cells and then allowed them for self-assembly before the sonication process (20 cycles) with 40% intensity and 10 seconds on/off the pulse). J aggregated ICG tarped biomimetic nanovesicles were dialyzed for 2 days against DI water (with dialysate changing every 12 h) and characterized further. The same process was also used to encapsulate the monomers of ICG (freshly prepared 0.01 mM in the aqueous medium at room temperature) in biomimetic nanovesicles.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAmphiphilic cargo encapsulation within biomimetic nanovesicles.\u003c/strong\u003e To understand the characteristics of lipid bilayer in cancer cell derived nanovesicles, J aggregates of lipophilic and red emissive Nile Red dye (0.01 mM in DMSO) were introduced in the mixture of small bilayers of ruptured ghosts\u0026rsquo; cells and then allowed them for self-assembly for overnight. This suspension was treated for the sonication process (20 cycles) with 40% intensity and 10 seconds on/off the pulse). To examine, the presence of surface biomarkers/proteins on the exterior surface of biomimetic nanovesicles, ICG dye (100 \u0026micro;L) was incubated with 500 \u0026micro;L of membrane nanovesicles (5 mg/mL) for overnight at ambient temperature followed by slow mixing. The obtained mixture was centrifuged (6000 rpm) for 5 minutes and dialyzed for 2 days (12 kD membrane) against DI water in the dark conditions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAqueous dispersibility, phototransduction, photosensitizing ability and photostability.\u003c/strong\u003e Various formulations of dye and drug tagged nanovesicles (100 \u0026micro;L) were mixed in an aqueous suspension (1 mL of PBS). These samples were tested for dispersibility at different time points (1st day to 30th day) followed digital photography measurements at room temperature conditions which were corroborated by spectroscopic measurements. For photostability measurements, the dye integrated biomimetic nanovesicles (0.2 mg/mL) were tested for spectroscopic study by measuring their emission properties at various time points (1 h to 30 days) with and without treating them in serum. Single membrane ghost nanovesicles accommodate a few hundred dye molecules which help in demonstrating better and controlled emission properties whereas free dye molecules lose their optical behavior due to light sensitivity. Fluorescence intensity and stability of dye tagged nanovesicles were recorded under 760 nm excitation. Time dependent (0\u0026ndash;5 minutes) phototransduction performance of designed optotheranostics (150 \u0026micro;L volume) was measured in an individual well of 96-well plate followed by NIR light irradiation (808 nm, 1 W power). Prior to phototransduction experiments, the water bath temperature was stabilized at 37.0\u0026deg;C. The temperature response was recorded in each 1 minute. For photosensitizing response of dye tagged nanovesicles, various concentrations (10\u0026ndash;200 \u0026micro;g/mL) of these vesicles were treated with NIR light. After complete NIR irradiation, these vesicles are stained with DCFDA (2\u0026prime;,7\u0026prime;-dichlorofluorescin diacetate) to measure the produced ROS.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn vitro\u003c/strong\u003e \u003cstrong\u003ebiocompatibility and cancer cell imaging.\u003c/strong\u003e L929 (5\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells per treatment) cell viability test was demonstrated at various concentrations (10\u0026ndash;500 \u0026micro;g/mL) of nanovesicles (BNVs), ICG encapsulated BNVs, Nile Red encapsulated BNVs, free ICG and Nile Red followed by MTT assay. In brief, normal cells were seeded and incubated for 24 h with 5% CO\u003csub\u003e2\u003c/sub\u003e in Dulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s Medium (DMEM Gibco, Carlsbad, CA, USA) supplemented with 10% Fetal Bovine Serum and penicillin/streptomycin at 37.0\u0026deg;C. After 24 h incubation, these cells were treated for further 18 h with 40 \u0026micro;L of each nanoparticle (10\u0026ndash;500 \u0026micro;g/mL) and then washed with PBS to remove excess particles from the treatment. To examine % cell viability, 20 \u0026micro;L of MTT dye was added to the above treatments and protected from light. Now, formazan crystals which were formed during the treatments were dissolved using 200 \u0026micro;L of DMSO. The optical absorbance was recorded using microplate reader (Tecan Infinite 200 PRO). Untreated cells were considered as control for calculating the % cell viability from the MTT assay.\u003c/p\u003e\n\u003cp\u003eFor cancer cell imaging and uptake studies, three different breast cancer cells (MCF-7, MDA-MB231 and 4T1, 5\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/well) were cultured in DMEM culture media supplemented with 10% Fetal Bovine Serum and penicillin/streptomycin followed by 24 h incubation in 5% CO\u003csub\u003e2\u003c/sub\u003e at 37.0\u0026deg;C. After the complete culture of cancer cells, 40 \u0026micro;L of dye (ICG and Nile Red) encapsulated nanovesicles were treated with breast cancer cells for 3 h. These treated cancer cells were washed with PBS several times to remove the unattached or free nanoparticles. Now, dye tagged nanovesicles (BNVs) treated cells were fixed with 4% paraformaldehyde and stained with 4, 6-diamidino-2-phenylindole (DAPI for cell nuclei). These treated and stained cells were mounted with the cover slip using 70% glycerol on a glass slide and then brought under fluorescence microscope to track the location of red dots (from dye tagged nanovesicles) and blue nuclei.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSolid tumor imaging and\u003c/strong\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003cstrong\u003ebio-distribution.\u003c/strong\u003e\u003cem\u003eIn vivo\u003c/em\u003e protocol in 6 weeks old female Balb/c mice (~\u0026thinsp;20 g weight) was approved by the Institutional Animal Ethical Committee. All experimental protocols on Balb/c female mice were approved by the Institutional Animal Ethical Committee (IAEC) of the National Centre for Cell Science, Pune, India (NCCS, Pune). Imaging studies were performed during anesthetized condition (50 \u0026micro;L dose with 1:4 of Xylazine and Ketamine for anesthesia). For solid tumor development, 5 x 10\u003csup\u003e5\u003c/sup\u003e 4T1 cells were injected subcutaneously into the mammary fat pad of female mice and kept for about 15 days of tumor growth observation which was measured using a caliper. On the 15th day of tumor growth, a single dose (10 mg/kg body weight containing 2.5\u0026times;10\u003csup\u003e13\u003c/sup\u003e nanoparticles/cm\u003csup\u003e3\u003c/sup\u003e) of three different nanoparticles (i. ICG J-aggregates biomimetic nanovesicles (interior), ii. ICG J-aggregates biomimetic nanovesicles (exterior) and iii. Nile Red dyes J-aggregates within lipid layers of biomimetic nanovesicles) were intravenously injected in mice. Nanoparticles administrated (10 mg/kg dose) tumor bearing animals were placed for whole body Near Infrared Fluorescence (NIRF) Imaging and scans were demonstrated for different time points (0.5 h, 1 h, 3 h, 6 h, 12 h and 24 h) under anesthetized conditions (50 \u0026micro;L dose with 1:4 of Xylazine and Ketamine for anesthesia). Fluorescence signals coming from nanoparticles were measured from whole body including tumor area and vital organs (heart, liver, spleen, and kidneys). Biodistribution measurements were evaluated by considering quantitative ROI values of the fluorescence intensity of the corresponding organs and tumors \u003cem\u003ein vivo\u003c/em\u003e. All imaging scans were done in anesthetized condition (50 \u0026micro;L dose of Xylazine and Ketamine for anesthesia) of animals (n\u0026thinsp;=\u0026thinsp;3). Several qualitative and quantitative analyses were done and compared with pre-injected animals (control) to evaluate the site-selective tumor imaging and biodistribution. Animals were euthanized under 5 minutes of CO\u003csub\u003e2\u003c/sub\u003e inhalation to collect tissues and tumor samples for \u003cem\u003eex-vivo\u003c/em\u003e and pathological examinations at different time points. Additionally, some animals were sacrificed after 24 h of injection and major organs were collected and tested for tissue pathological examinations (hematoxylin and eosin, H\u0026amp;E) which were conducted for \u003cem\u003ein vivo\u003c/em\u003e toxicity and to investigate the tissue injuries of injected nanoparticles.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn vivo\u003c/strong\u003e \u003cstrong\u003etoxicity and clearance measurements.\u003c/strong\u003e All treated mice were sacrificed after injecting nanoparticles and collected major organs and tumors were made for histopathological examinations (Hematoxylin and Eosin, H\u0026amp;E) to study the tissue damage. Additionally, body weight measurements, health behavior, hair loss, eschars and inflammation on animal\u0026rsquo;s body of treated animals were also examined to ensure the adverse effect of injected nanoparticles.\u003c/p\u003e\n\u003ch2\u003eDye per nanoparticle and effective brightness calculation\u003c/h2\u003e\n\u003cp\u003eBefore calculating the nanoparticles per cm\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, dye per nanoparticles and their relative brightness, concentration of engineered dye tagged nanovesicles was measured after drying (48\u0026ndash;50\u0026deg;C) a known volume (150 \u0026micro;L) of nanoparticles (60 nm in diameter) followed by dry residue of used solvent as a baseline. The mass of the particle per unit volume of the solution was 0.3 mg/mL with 1.36 x 10\u003csup\u003e\u0026minus;\u0026thinsp;13\u003c/sup\u003e mg weight of one nanoparticle. Further, we have calculated the number of particles (7.3 x 10\u003csup\u003e11\u003c/sup\u003e nanoparticles/cm\u003csup\u003e3\u003c/sup\u003e) in the stock solution using the mass density (1210 mg/cm\u003csup\u003e3\u003c/sup\u003e) and diameter of nanovesicles (60 nm). Spectroscopic methods were applied to estimate the number of dye molecules per cm\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e (5.74 x 10\u003csup\u003e13\u003c/sup\u003e dye molecules/ cm\u003csup\u003e3\u003c/sup\u003e) and dye per nanoparticles (786) considering extinction coefficients 2.621 x 10\u003csup\u003e5\u003c/sup\u003e M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at 780 nm and Beer\u0026ndash;Lambert law. Importantly, the same concentration of free dye was considered and absorption of free nanovesicles was subtracted for estimating these numbers including relative brightness (737). We have measured the fluorescence which was coming from the solution of ICG encapsulated nanovesicles (known concentration) and the reference dyes ICG. To estimate the numbers of cargo molecules loaded within a single nanovesicle viz., BNV, we need to estimate the mass of a single nanovesicle considering radius (r) and using the equations:\u003c/p\u003e\n\u003cdiv id=\"Eque\" class=\"Equation\"\u003e\n \u003cdiv class=\"mathdisplay\" id=\"FileID_Eque\" name=\"EquationSource\"\u003e\u003cimg 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\"\u003e\u003cbr\u003e\u003c/div\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eStatistics\u003c/strong\u003e. For statistical analysis, all experiments were performed in triplicate. All data were analyzed and plotted using OriginPro 8. Significant observations between different groups were calculated by t-test.\u003c/p\u003e\n\u003cp\u003eASSOCIATED CONTENT\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupporting Information.\u003c/strong\u003e cryo-TEM images, DLS measurement, UV-Vis and emission spectra, equations for drug loading efficiency, ultra brightness and photostability data, Zeta potential measurement, digital photographs, release kinetics data, and experimental setup, cell imaging, \u003cem\u003ein vivo\u003c/em\u003e distribution and whole-body scans.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eNIR, Near-Infrared,\u0026nbsp;\u0026nbsp;\u0026nbsp;density,\u0026nbsp;\u0026nbsp;\u0026nbsp;concentration, DLS, dynamic light scattering, \u0026lambda;\u003csub\u003eabs/em\u003c/sub\u003e represent absorption/emission, A absorbance, \u0026epsilon; extinction coefficient, TEM, transmission electron microscopy, FLBNV fluorescence from BNV, CBNV concentration of BNVs, FL Dye fluorescence from organic dyes, C Dye concentration of organic dyes, r radius of single BNV, MESF units (Molecules of Equivalent Soluble Fluorochrome), ICG Indocyanine green was named as ICG.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAuthor Contributions\u003c/p\u003e\n\u003cp\u003eR.P. has designed the project and has done major experiments for engineering nanoparticles, and their spectroscopic characterization. K.P. contributed to providing cells, washing them, and cell imaging studies. M.T. has done some preparation for nanoparticles and has performed cryo-TEM of prepared nanovesicles. J.B. has helped for cryo-TEM imaging. M.G. P.B. and G.C.K. have done \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e studies. V.K.D has provided the cells and related facility. P.C. and R.K. have helped in the manuscript preparation. S.K. has helped in in-vivo analysis. All authors have contributed to writing and revising the draft and have approved the final version of the manuscript. \u003csup\u003e$\u003c/sup\u003eThese authors have contributed equally to this project.\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eR.P. holds patents related to lipid, gold and silica nanoparticles. All other authors declare no competing financial interest.\u003c/p\u003e\n\u003cp\u003eACKNOWLEDGMENT\u003c/p\u003e\n\u003cp\u003eR.P. thanks the director and the School of Biochemical Engineering IIT (BHU) Varanasi for providing the necessary facilities and support. This work was supported by the Departmental Instrumentation and Laboratory facilities. The authors would like to thank the Central Discovery Centre (CDC) and SATHI, IIT (BHU) for cell-imaging studies. We thank Sahu Bio Tech Services, Kirkatwadi, Pune, Maharashtra, India, for laser support and suggestions during the project.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eReprints and permissions information is available at http://www.nature.com/reprints\u003c/p\u003e\n\u003cp\u003eData availability\u003c/p\u003e\n\u003cp\u003eData will be made available on request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWeng, J.; Wang, Y.; Zhang, Y.; Ye, D. 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C.; Xia, J.; Lovell, J. F.; Conde, J. Biomimetic Bright Optotheranostics for Metastasis Monitoring and Multimodal Image-Guided Breast Cancer Therapeutics. J. Control. Release 2024, \u003cem\u003e367\u003c/em\u003e (January), 300\u0026ndash;315. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jconrel.2024.01.056\u003c/span\u003e\u003cspan address=\"10.1016/j.jconrel.2024.01.056\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"npj-imaging","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [npj Imaging](https://www.nature.com/npjimaging)","snPcode":"44303","submissionUrl":"https://submission.springernature.com/new-submission/44303/3","title":"npj Imaging","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Biomimetic, Ultrabright, Nanoparticles, In vivo Imaging, Solid Tumor ","lastPublishedDoi":"10.21203/rs.3.rs-4616433/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4616433/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOptically active biomimetic ghosts nanovesicles are highly potent as imaging agents for site-selective solid tumor imaging with deep tissue visualization. However, reported systems are limited with poor brightness and photostability with NIR absorption and emission. Herein, cancer cell membrane derived biomimetic ghost nanovesicles (~60 nm) have been engineered with amphiphilic dyes aggregates for site-selective solid tumor imaging in pre-clinical models. Entrapped dye aggregates within biomimetic ghost nanovesicles (BNVs, 505 to 828 dye molecules/vesicle) exhibit promising fluorescence and photostability (up to 30 days) showing ultra-brightness (778 MESF) with promising tumor fluorescence signals (760 nm excitation) compared to free dye molecules and dye aggregates. Dye aggregates-BNVs exhibit significantly different imaging response than amphiphilic monomers-BNVs. Lipophilic and amphiphilic structural layers and surface biomarkers of ghost nanovesicles are examined through physicochemical measurements, corroborated with cargo release kinetics. Controlled body weight, long time survival and histopathology examinations ensure the \u003cem\u003ein vivo\u003c/em\u003e biocompatibility of these intravenously administrated biomimetic imaging agents. Our findings suggest that these ghosts nanovesicles mimic the biological characteristics of native cells, enabling them to evade immune clearance.\u003c/p\u003e","manuscriptTitle":"NIR Emissive Biomimetic Ghost Nanovesicles for Site-Selective Solid Tumor Imaging","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-09 16:33:57","doi":"10.21203/rs.3.rs-4616433/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorAssigned","content":"","date":"2024-07-09T14:57:35+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-08T09:26:18+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Imaging","date":"2024-06-21T09:32:01+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-imaging","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [npj Imaging](https://www.nature.com/npjimaging)","snPcode":"44303","submissionUrl":"https://submission.springernature.com/new-submission/44303/3","title":"npj Imaging","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f3e927ce-e476-45e0-8c3a-9afcbcf89751","owner":[],"postedDate":"August 9th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":34354688,"name":"Biological sciences/Biotechnology"},{"id":34354689,"name":"Physical sciences/Nanoscience and technology"},{"id":34354690,"name":"Physical sciences/Optics and photonics"}],"tags":[],"updatedAt":"2024-08-09T16:33:58+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-09 16:33:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4616433","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4616433","identity":"rs-4616433","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}