Trastuzumab herceptin

Human HER-2-positive breast cancer cell lines SKBR-3 and BT474 were purchased from American Type Culture Collection (Manassas, USA) and maintained in Dulbecco’s modified Eagle’s medium (HyClone Lab., Inc., Logan, UT) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich, St. Louis, MO, USA), 100 U/ml penicillin, and 100 μg/ml streptomycin (Life Technologies, Grand Island, NY, USA) in humidified air at 37 °C with 5% CO₂. The cell lines were authenticated by short tandem repeat profiling. Trastuzumab (Herceptin) was purchased from Roche (Basel, Switzerland) and was used by dissolving in phosphate-buffered saline (PBS). The SKBR-3 and BT474 cells resistant to trastuzumab treatment (named as SKBR-3-TR and BT474-TR, respectively) were built by establishing xenografts followed by four courses of trastuzumab treatment as previously described^{24 (link)}.

Hydroxypropyl Beta cyclodextrin (HPβCD) was obtained from Acros (Belgium) and Beta cyclodextrin (βCD) was purchased from Sigma (USA). Trehalose dehydrate, potassium phosphate dibasic and disodium sulfate were acquired from Merck (Germany). Trastuzumab (Herceptin®) was purchased from Roche Ltd (Hungary) and the chemicals were from sigma (USA). Human IgG₁ (with molecular weight of about 150 KD) was supplied by Kedrion (Italy). Prior to each investigation, low molecular weight additive of antibody solution was removed by dialysis with deionized water (cut off: 15 kDa).

PLGA-PEG-PLGA (MW: PEG = 1500 Da, MPLGA: MPEG = 3:1) was purchased from Shandong Daigang Inc. (Jinan, China). Collagenase-I was supplied by Invitrogen (Carlsbad, CA) and trastuzumab (Herceptin) was obtained from Roche (Basel, Switzerland). Cy7-NHS ester was obtained from GE Company (Boston, MA). Anti-CD31, anti-CD68, and anti-collagen antibodies were purchased from Abcam (Cambridge, UK). RPMI-1640, penicillin/streptomycin, and trypsin were purchased from M&C Gene Technology (Beijing, China). Fetal bovine serum (FBS) was from Gibco (Invitrogen, Carlsbad, CA). BT474 cells were from China Center for Type Culture Collection (Wuhan, China).

The hamster infection model of SARS-CoV-2 has been described before (Boudewijns et al., 2020 (link); Kaptein et al., 2020 (link)). For infection, animals were anesthetized with ketamine/xylazine/atropine and inoculated intranasally with 50 μL containing either 2×10⁶ TCID50 SARS-CoV-2 (ancestral Wuhan strain), 1×10⁴ TCID50 (Beta B.1.351), or 1×10⁴ TCID50 (Delta B.1.617.2). Antibody protein treatments (anti-SARS-CoV-2 mAbs or human IgG1 isotype control Trastuzumab/Herceptin® (Roche)) were initiated 24 h post infection by intraperitoneal injection. Intramuscular pDNA electroporation was done at d-10, d-7 and d-5 infection. Hamsters were monitored for appearance, behavior, and weight. At day 4 post-infection, animals were euthanized by intraperitoneal injection of 500 μL Dolethal (200 mg/mL sodium pentobarbital, Vétoquinol SA). Lungs were collected and viral RNA and infectious virus were quantified by RT-qPCR and end-point virus titration, respectively. Blood samples were collected at end-point for pharmacokinetic analysis. No randomization methods were used and confounders were not controlled, though all caretakers and technicians were blinded to group allocation in the animal facility and to sample numbers for analysis (qPCR, titration, and histology).

Poly(allylamine hydrochloride) (PAH) with Mw ~15,000, poly(sodium 4-styrenesulfonate) (PSS) with Mw ~70,000, and polyacrylic acid (PAA) with Mw ~15,000, and bovine serum albumin (BSA) were purchased from Sigma-Aldrich, USA. Sodium carbonate, calcium chloride, and ethylenediaminetetraacetic acid disodium salt dehydrate (EDTA) were obtained from Sigma-Aldrich, Germany. Carboxyl- and sulfhydryl-terminated derivative of 12-unit polyethyleneglycol CT(PEG)₁₂, N-hydroxysulfosuccinimide (sulfo-NHS), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC), sulfosuccinimidyl and 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) were purchased from Thermo Fisher Scientific, USA. CdSe/ZnS (core/shell) QDs with a fluorescence maximum at 590 nm were kindly provided by Dr. P. Samokhvalov (LNBE, MEPhI, Moscow, Russia). The humanized monoclonal anti-HER2 antibody, Trastuzumab (Herceptin^®), were obtained from Roche, Switzerland.
All the other reagents were of analytical grade and obtained from Sigma-Aldrich, USA. All working polymer and buffer solutions were prepared using MilliQ water (18.2 mΩ·cm) obtained by means of a Direct-Q water purification system (Millipore, France) and additionally filtered through sterile filters with a pore size of 0.22 μm (Millipore, France).