In vivo imaging and biodistribution of near infrared dye loaded brain-metastatic-breast-cancer-cell-membrane coated polymeric nanoparticles

The hydrodynamic size of the NP and CCNP were 43 ± 5 nm and 70 ± 4 nm, and the surface charge of the nanoformulations was −10 ± 2 mV and −20 ± 4 mV respectively (figures 1(A) and (B)). The size of our nanoformulation is smaller than previously reported [22]. The size of the IR 780 loaded CCNP were 55 ± 5 nm and 76 ± 7 nm respectively (figure 1(A)). The optimum size and surface charge of nanoparticles which can cross the BBB effectively are smaller than 125 nm and lower than −30 mV respectively [23]. Hence, the size and surface charge of the nanoparticles were optimized to make it below 100 nm and lower than −30 mV to cross the BBB to reach the brain (ESI table 1). CCNPs were stable for two weeks at 4 °C in PBS (ESI 2).

The UV–vis spectra showed the absorption peak of the NP and CCNP were located at 780 nm, which correlated with the free-dye absorption peak of IR 780-I (figure 1(C)). Our results were similar to the previously reported ICG loaded ICNP nanoparticles [24]. The absorption spectra of the DOX-loaded NP and CCNP were identical. The blank NP and CCNP, which did not contain any DOX, did not show any fluorescence intensity, as expected. Membrane coating on DOX loaded NP did not affect the fluorescence property of the nanoformulation. The loading efficiency of DOX into the NP was around 10% ± 2%.

AFM analysis was performed to check the shape and distribution pattern of the NP and CCNP (figure 2). AFM image and average size distribution analysis revealed that the NP and CCNP were spherical and uniform in size.

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Protein stability of the CCNP measured by SDS-PAGE showed that both CCNP and cell membrane fraction had similar protein profiles compared to the MDA-MB-831 whole cell lysate (ESI figure 3). Brain metastatic cancer membrane isolated by ultracentrifugation was coated on the NP by extrusion. Brain metastatic breast cancer membrane form a thin film/layer of the NP upon extrusion making the core–shell hybrid nanoparticle. NP acts as core whereas brain metastatic breast cancer membrane represent the shell structure. The membrane protein present on the cancer membrane get translocated on the NP, thus helping in escaping the immune and precise targeting.

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Cellular uptake of the nanoparticles by MDA-MB-831 cells was measured by incubating cells with various Nile-red loaded nanoformulations and visualized under confocal laser scanning microscope (CLSM) (figure 3). The CCNP showed better cellular uptake compared to NP alone. To illustrate the membrane coating, a green fluorescent dye NHS-Fluorescin was tagged to extracted membrane before coating. Our results were similar to the previously reported results [16] of the CCNP in the cytoplasm and the membrane coating was intact during uptake.

The cytotoxicity of DOX-loaded NPs and CCNPs was analyzed by MTT assay (ESI figure 4). Cells were treated with equal concentrations of DOX-loaded formulations for 24 h. The DOX-loaded CCNPs proved to be more cytotoxic compared to the DOX-loaded uncoated NP owing to cancer membrane coating. Dox-loaded CCNP showed an approximately 2-fold increase in cytotoxicity compared to the control (P < 0.001). The cytotoxic effect of Dox-loaded CCNP was significantly higher compared to the NP (P < 0.05). Our results were similar to previously reported cancer-membrane based nanoformulations [25].

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To investigate the feasibility of these nanoformulations for in vivo fluorescent imaging and to determine their ability to cross the BBB, biodistribution studies were performed in athymic nude mice. IR 780-I loaded nanoformulations (0.1 mg ml⁻¹ gm⁻¹ weight) in HBSS were injected into six to ten week old mice via tail vein and monitored for real-time whole-body imaging. The mice were imaged at 24, 48, 72, 96 and 120 h using an IVIS live imaging system to determine the distribution pattern of the NPs (figure 4).

At 24 h the total intensity of the organs from the animals treated with CCNPs was slightly higher than that of uncoated NP and then started reducing thereafter till 120 h. Our results indicate that coating the NP with the cancer cell membrane has further increased the retention time compared to the uncoated NP.

Biodistribution profile was examined in vital organs (brain, hearts, lungs, liver, kidney, and spleen) at 24 and 48 h (figure 5). In vivo biodistribution studies showed higher accumulation and retention of CCNP in the brain, lungs and spleen till 48 h. The intensity of CCNP fluorescence in brain was 2-fold higher compared to NP fluorescence till 48 h (P > 0.001). whereas, the intensity of CCNP fluorescence in the lungs was near to 2-fold higher compared to NP fluorescence (P < 0.05). In contrast to lungs, the fluorescence intensity of the CCNP in the spleen was similar to the fluorescence intensity of NP at 24 h (P < 0.05), while CCNP intensity was increased by 2.5–3-fold (P < 0.001) from 24 to 48 h.

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The high intensity in lungs and spleen indicate the extended blood circulation of these nanoformulations due to its stealth properties. However, the high intensity of NP in Lungs and spleen infers the slow clearance of the NP by the reticuloendothelial-system. ICNP nanoparticles showed similar results by escaping the liver and kidney clearance [26]. The nanoparticles in the range of 15–200 nm are cleared majorly by Kupffer cells in the liver and splenic macrophage cells [21]. Longer circulation and stealth properties have been found suitable for the brain targeting. The increase in the fluorescent intensity of CCNP in brain compared to NP may be attributed to high cellular uptake by brain cells. This is most likely due to the cancer membrane coating derived from the brain metastatic cells. One of the possible reasons could be the attachment of CCNP to the endothelial cell due to the presence of brain metastatic cancer membrane. The vascular endothelial growth factor (VEGF) receptor present on the cancer membrane further helps in BBB pentation. Reactive astrocytes surround the brain metastatic tumor membrane facilitates in further movement in association with VEGF [27]. Because these membranes contain proteins which enable the cells to breach the BBB and metastasize to the brain, coating the nanoparticles with these membranes may confer stealth abilities on the nanoparticles. PEG coating has also been shown to enhance the brain cellular uptake by providing stealth properties in circulation [28]. There were no statistically significant differences in both the time points 24 and 48 h. One of the probable reasons could be the slow movement of fluid in the brain compared to the cellular uptake of these nanoformulations due to stealth properties. The rate of clearance of the NP depends on the movement of the NP through the interstitial fluid to the brain capillaries from where it gets eliminated [28]. Thus, in vivo imaging data inferred the suitability of CCNP for targeting brain metastasis of breast cancer. A further study with drug is required to validate this. Enhanced retention in the brain could be desirable for controlled drug release. On the other hand, enhanced circulation may lead to toxicity in healthy tissues. To demonstrate the biocompatibility of these nanoformulations histopathological evaluation of the extracted organs (brain, heart, lungs, liver, kidney, and spleen) were performed (ESI figure 5). No major perturbations to normal organ tissue structure was observed. Hence, CCNP could be safe and effective nanocarrier for cancer theragnostic of brain metastatic breast cancer.

Nanoparticles were prepared using a nanoprecipitation method [15]. Briefly, 4 mg of mPEG-PLGA was dissolved in Acetonitrile (ACN) and added dropwise into water under constant stirring. The nanoparticles were purified and concentrated using 10 kD MWCO Amicon filters. The concentrated nanoparticles were re-suspended in water and stored at 4 °C until further use. To prepare dye/drug-loaded nanoparticles, dye/drug was dissolved in the organic phase and emulsified similar to the preparation of the nanoparticles.

Cell membranes from the brain metastatic breast cancer cell line MDA-MB-831 were extracted using a previously reported method [16]. Briefly, cancer cells were grown in T-175 flask till it reached 70%–80% of confluency and harvested by detaching in 2 mM EDTA solution (Sigma-Aldrich) in PBS. Cells were washed thrice with PBS at 500 g for 5 min g is the unit of the relative centrifugal force represented as time gravity.

Cells were further lysed in hypotonic solution (20 mM Tris-HCl pH 7.5 Sigma-Aldrich,10 mM KCl Sigma-Aldrich, 2 mM MgCl2 Sigma-Aldrich and 1 mM EDTA free protease cocktail (Pierce)) and homogenized using Dounce homogenizer. The lysate was centrifuged at 3500 g for 5 min. The supernatant was collected and centrifuged again at 20 000 g for 20. The pellet was discarded, and the supernatant was centrifuged at 100 000 g for 20 min. The supernatant was discarded, and the pellet containing cancer cell membrane was washed with 10 mM Tris-HCl and 1 mM EDTA. The cancer cell membrane was freshly used for nanoparticles coating.

The MDA-MB-831 cell membrane was extruded through an Avanti extrusion chamber using 400 nm polycarbonate membranes. The extruded membrane was mixed with polymeric nanoparticle (1:1) and extruded again through a 200 nm polycarbonate membrane to obtain the final cancer membrane coated NP. For confocal microscopic experiment, N-hydroxysuccinimide-Fluorescein isothiocyanate tagged cancer membrane was coated with nile-red dye loaded nanoparticles. Briefly, NHS-fluorescein was dissolved in small amount of DMSO (Dimethyl sulfoxide) and incubated with cancer membrane overnight with mild intermittent shaking at 4 °C. Untagged NHS-fluorescein was washed by 3.5 kD dialysis membrane against the HBSS for 5–6 h. The dialysis buffer was changed every hour and checked for FITC fluorescence until no significant trace was observed. The NHS FITC tagged cancer membrane was coated over the nanoparticles by passing it through the syringes 11 times similar to the extrusion process and analyzed by confocal microscopy (Zeiss microscopy, Carl Zeiss, GmbH) for in vitro cellular uptake studies.

The protein contents in the cell lysate, isolated membrane and CCNP were determined using a Bradford Assay. Equal concentrations (10 μg) of protein were separated by electrophoresis on a 4%–12%Bis-Tris SDS-PAGE gel (Thermo Fisher). The gel was washed and stained with Coomassie blue. The gel was destained and visualized on an imaging system (Alpha Innotech fluor Chem, US).

The particle size and surface charge of the freshly prepared nanoparticles were characterized by zeta sizer (Malvern Instruments, US). The absorbance of drug/dye-loaded nanoparticles was characterized by UV–vis spectroscopy (Thermo Scientific US). The size and morphology of the nanoparticles were characterized by atomic fluorescence microscopy (AFM) (NT-MDT NTEGRA, US). All experiments were performed in triplicate. The stability study was performed by measuring the hydrodynamic size in PBS. The size of the particles after synthesis and after two weeks was measured by DLS.

MDA-MB-831 cells were cultured in DMEM high-glucose media (Hyclone Labpratory Inc. US) containing 10% FBS with penicillin, streptomycin, and Amphotericin B (ThermoFisher) [29]. Cells were verified by PCR to be free of mycoplasma.

MDA-MB-831 cells were seeded on coverslips at a density of 2 × 10⁴ cells/well of a 12 well plate. The media was replaced with fresh serum-free DMEM media supplemented with 200 μl of loaded nanoparticles (200 μg) and cultured for an additional two hours. For cell labeling, Nile red dye was loaded into the NPs and CCNPs. To detect the membrane coating, cell membrane vesicles were labeled with NHS-fluorescein (Pierce) as previously reported. The cells were washed thrice with PBS, fixed with 4% paraformaldehyde and mounted with mounting media containing DAPI. The cells were visualized under CLSM. The images were processed post-acquisition by Zen software (Carl Zeiss, GmbH).

MDA-MB-831 cells were seeded into 96 well plates. The cells were grown for 24 h at 37 °C with 5% CO2. Doxorubicin-loaded NPs and CCNPs were mixed in fresh serum-free media and added to the cells. Following a twenty-four-hour incubation, 10 μl of 10.5 mg ml⁻¹ of MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) was added to the cells. After 2 h, the media was removed, and formazan crystals were dissolved in 100 μl of DMSO and read on microplate reader preset with MTT reading parameters.

Female athymic nude mice Fox n1/nu (four to five weeks old) were purchased from Envigo, US. All animal studies were performed in compliance with a protocol approved by Institutional Animal Care and Use Committee. The IR 780-I dye-loaded nanoparticles in PBS were injected into the mice via tail vein injection. In vivo imaging at different time points were performed using a Caliper Lumina XR system (λex 745 nm and λem ICG/780 nm).

The mice were sacrificed at the end of the whole-body imaging, and organs (brain, heart, lungs, liver, spleen, and kidney) were extracted, washed with PBS and fixed in 4% paraformaldehyde in PBS solution for 24 h at 4 °C. The images were captured immediately by Lumina XR system (λex 745 nm and λem ICG/780 nm), Perkin Elmer US. For histological sections, the organs were fixed, washed thrice with 70% alcohol and embedded in paraffin. The images of H and E stained slides were captured using a Leica microscope, Germany.

All experimental data were analyzed as mean ± SD. The standard contingency table and analysis of variance techniques (t-test) were used to determine differences in fluorescence distributions within the various organs (P < 0.05).