Biomorphic Engineering of Multifunctional Polylactide Stomatocytes toward Therapeutic Nano‐Red Blood Cells

Abstract Morphologically discrete nanoarchitectures, which mimic the structural complexity of biological systems, are an increasingly popular design paradigm in the development of new nanomedical technologies. Herein, engineered polymeric stomatocytes are presented as a structural and functional mimic of red blood cells (RBCs) with multifunctional therapeutic features. Stomatocytes, comprising biodegradable poly(ethylene glycol)‐block‐poly(D,L‐lactide), possess an oblate‐like morphology reminiscent of RBCs. This unique dual‐compartmentalized structure is augmented via encapsulation of multifunctional cargo (oxygen‐binding hemoglobin and the photosensitizer chlorin e6). Furthermore, stomatocytes are decorated with a cell membrane isolated from erythrocytes to ensure that the surface characteristics matched those of RBCs. In vivo biodistribution data reveal that both the uncoated and coated nano‐RBCs have long circulation times in mice, with the membrane‐coated ones outperforming the uncoated stomatoctyes. The capacity of nano‐RBCs to transport oxygen and create oxygen radicals upon exposure to light is effectively explored toward photodynamic therapy, using 2D and 3D tumor models; addressing the challenge presented by cancer‐induced hypoxia. The morphological and functional control demonstrated by this synthetic nanosystem, coupled with indications of therapeutic efficacy, constitutes a highly promising platform for future clinical application.


Dynamic light scattering measurements (DLS)
DLS measurements were performed by using a Malvern Instruments Zetasizer (model Nano ZSP).
Zetasizer software was used to process and analyze the data.

Nuclear magnetic resonance spectroscopy (NMR)
Proton nuclear magnetic resonance measurements were performed on a Bruker 400 Ultrashield TM spectrometer equipped with a Bruker SampleCase autosampler, using CDCl 3 as a solvent and TMS as internal standard.

Differential scanning calorimetry (DSC)
DSC measurements were conducted using a TA Instruments Multicell DSC. By scanning from -20 o C up to 80 o C at 5 o C per minute, the Tg value was recorded from the second heating run.

Gel permeation chromatography (GPC)
Molecular weights of the block polymer were determined by using a Prominence GPC system S PL D P L fferential refractive index detector. THF was used as an eluent with a flow rate of 1 mL per minute. Polystyrene strandards (580~377400 g mol -1 ) were used for calibration.

Confocal laser scanning microscopy (CLSM)
Fluorescence images were observed and captured by using CLSM (Zeiss LSM510 META NLO, and Leica TCS SP5X).

UV-vis spectroscopy
Drug loading efficiency, BCA protein assay, as well as the generation of singlet oxygen were characterized by UV-vis spectroscopy (V-650, JASCO).

Flow cytometry
Cell uptake and cell viability were measured by flow cytometry (BD Biosciences, USA).

In-Vivo imaging systems
Biodistribution of nano-RBCs in vivo was measured by In-Vivo imaging systems (FX Pro, KODAK) at designed time points. with DCM, extracted with KHSO 4 (2x), water and brine. The solution was dried with Na 2 SO 4 , filtered and concentrated in vacuo yielding a yellowish oil. To remove the Boc protective group, the polymer was dissolved in 5 mL of 4 M HCl in dioxane for 1 hour. The reaction mixture was concentrated in vacuo, dissolved in dioxane and freeze-dried, yielding a white powder (83%). The obtained amphiphilic polymer was characterized using DSC and SEC. For PEG 44 -PDLLA 120 and amino-PEG 44 -PDLLA 120 , the Tg value was 21 o C, and the PDI was 1.07. The relative M n s compared to PS standards obtained from GPC were 29.6 and 19.8 kg mol -1 , respectively. All the products were stored at -20 o C under argon until use.

Fabrication of stomatocytes and loading with hemoglobin (Hb)/Chlorin e6 (Ce6)
PEG 44 -PDLLA 120 and amino-PEG 44 -PDLLA 120 (9:1 w/w, 20 mg) were dissolved in 2 mL of mixed organic solvent (THF : dioxane = 1:4 v/v) in a 15 mL vial. Then a magnetic stirring bar was added to the solution and the vial was sealed with a rubber septum. The mixed solution was stirred for at least 30 min before adding 2 mL of ultrapure MilliQ water via a syringe pump (1 mL h -1 ). Afterwards, the resulting cloudy solution was transferred into a prehydrated dialysis bag (12-14 kDa, 2 mL cm -1 ) and dialyzed against a pre-cooled NaCl solution (50 mM Hb (628 nm) was measured by UV-vis spectroscopy. [2] The drug loading efficiency (DLE) is defined as the ratio of weight of drug loaded to the weight of drug in the feed. [3] The DLE of Ce6 and Hb were 44.76% and 97.50%, respectively. Furthermore, Hb loading was characterized by SDS-PAGE. [4]

Preparation and characterization of RBC vesicles, and RBC-derived vesicle modified stomatocytes
The RBCs were isolated from peripheral blood of healthy mice (Balb/c, male) following published protocols. [5] Briefly, blood was centrifuged for 5 min (800×g, 4 o C) and then washed three times with ice cold PBS buffer (pH 7.4, 1×). Hemolysis was obtained by adding the hypotonic solution and then storing the above-mentioned solution overnight at 4 o C. After that, the blood samples were treated on an ice bath for 20 min, followed by washing with PBS buffer and sonication. The obtained RBCderived vesicles were stored at 4 o C for further use. The RBCs and RBC-derived vesicles were characterized by SEM and CLSM. Erythrocyte membrane coated stomatocytes were prepared according to a published method. [5] Stomatocytes were mixed with the RBC-derived vesicles and co-cultured for 4 h under gentle shaking at 4 o C. Free RBC-derived vesicles were removed by centrifugation at 10000 rpm for 1 min. For observation by CLSM, RBC-derived vesicles and erythrocyte membrane coated stomatocytes were stained with the Alexa Fluor TM 488 conjugate of wheat germ agglutinin. The surface charge changes of stomatocytes before and after coating were measured by Zetasizer. Surface protein content of stomatocytes before and after coating with the erythrocyte membrane were measured by a BCA protein assay kit. Additionally, the yield of membrane coating process was measured by flow cytometry, which is 91.51%. Here, the fluorescent signal from Ce6 was used to count the whole number of stomatocytes, whilst cell membrane fluorescent dye (Alexa Fluor TM 488 conjugate of wheat germ agglutinin) was detected to count the membrane coating stomatocytes.

Evaluation of the generation of singlet oxygen ( 1 O 2 )
9,10-Anthracenediyl-bis(methylene)dimalonic acid (ABDA) was used as an indicator to assess the generation of 1 O 2 , according to a chemical oxidation method. [6] Stomatocytes (1.5 mL, 0.25 mg mL -1 ) ABDA DMSO L, 4 mg mL -1 ), followed by illumination with a 660 nm laser (BeamQ Lasers). The absorbance intensity of ABDA at 400 nm as a function of time was recorded by UV-Vis spectroscopy.

Cell culture
Mice embryonic fibroblast cells (NIH/3T3), human cervical cancer cells (HeLa), murine breast carcinoma cell line (4T1), liver hepatocellular carcinoma (HepG2) and mice macrophage cells (RAW 264.7) were cultured in cell culture medium supplemented with 10% FBS and 1% penicillinstreptomycin at 37 o C in the cell incubator (ThermoFisher Scientific) with an atmosphere of 5% CO 2 and 70% humidity. For NIH/3T3, HeLa, and 4T1, DMEM was used as cell culture medium. For RAW 264.7, RPMI 1640 medium was used.

Uptake efficacy
The uptake efficacy of stomatocytes before and after coating with the erythrocyte membrane towards RAW 264.7 was measured by flow cytometry. Briefly, nano-RBCs (0.25 mg mL -1 ) were incubated with RAW 264.7 for 0 h, 6 h, and 12 h. Non-internalized stomatocytes were removed through washing the cells with PBS three times. The fluorescent signal was detected by flow cytometry. The same method was user to determine the cellular uptake of HeLa, NIH/3T3 and HepG2 towards the nano-RBCs.

In vivo fluorescence imaging
Female BALB/c nude mice (6 weeks, body weight ~18 g) were obtained by Department of Experimental Animals, Institute of Process Engineering, Chinese Academy of Sciences (Beijing, China).
After acclimatization for 5 days, the mice were randomaly divided into three groups, and injected intrave L -RBCs, and uncoated stomatocytes (1 mg mL -1 ,

PBS H T L PBS H H
infrared fluorescent dye, Cy7 was loaded into the stomatocytes for in vivo fluorescence imaging. The biodistribution of nano-RBCs was observed by In-Vivo imaging system at designed time points. All animal experiments were conducted under the guidelines and approved by the local ethics committe.

Cell viability
NIH/3T3 and HeLa cells were used for cytotoxicity studies. Cells were diverted to 96-well plates using a standard trypsin-based technique with a final concentration of 5×10 4 cells per mL. When the cell density reached 90%, different samples at a range of concentrations (0, 0.05, 0.1, 0.15, 0.2, and 0.25 mg mL -1 ) were added. Then a standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to evaluate cell viability in the presence of the different samples.

In vitro evaluation of photodynamic therapy (PDT)
H L -Slide 8 wells (Ibidi) and cultured in DMEM cell culture medium containing 10% FBS and 1% penicillin-streptomycin at 37 o C, 5% CO 2 and 70% humidity. To optimize the culture time, stomatocyte samples were cultured with cells for 0 h, 2 h, 4 h, and 6 h, and then measured by CLSM (Leica TCS SP5X) and flow cytometry. To evaluate the therapeutic efficacy, 0.25 mg mL -1 samples were co-cultured with HeLa cells for 6 h, followed by irradiation with a 660 nm laser (0.1 W cm -1 ) for 5 min, after washing the cells with HBSS to remove stomatocytes that were not taken up by the cells. The cells were then cultured for another 12 h in the cell incubator, and analyzed by CLSM after live/dead fluorescent staining. The cell viability after PDT treatment was quantified with an MTT assay and by flow cytometry.

3D multi-cellular spheroid (MCS) tumor model
3D MCS tumor models were produced by co-culturing NIH/3T3 and 4T1 (5:1) cells, according to previously published protocols with slight modifications. [7] Cells were seeded in agarose coated 96well plates. 0.15 g of agarose was added to 10 mL of low glucose DMEM cell culture medium (1.5% wt/vol) in an appropriate beaker, followed by sealing with an aluminum foil/lid. After autoclaving (120 o C, 20 min), the agarose solution was transferred to a 96-L under sterile conditions. Then a concave surface was formed by the solidification of the agarose.
Next, NIH/3T3 and 4T1 cells with a L, 6×10 4 cells mL -1 in high glucose DMEM cell culture medium) were co-seeded in the above-mentioned 96-wells plate, and were then cultured in the incubator with an atmosphere of 5% CO 2 and 70% humidity for 4 days for the formation of MCSs.
For evaluation of the PDT efficacy, MCSs were divided into five groups and cultured with different stomatocyte samples for 6 h. Cold PBS was then used to wash the MCS three times. Subsequently, the MCSs were irradiated with a 660 nm laser for 5 min (660 nm, 0.1 W cm -2 ) and cultured for another 12 h. Calcein-AM and PI were used to co-stain the MSCs for assessing the cell viability with CLSM. The corresponding integrated fluorescent intensity of each channel was further analyzed by ImageJ.