Synthesis and Application of Novel Pertuzumab and Trastuzumab loaded Polycaprolactone Microspheres for the Inhibition of HER2 Receptors in Breast Cancer

By Keira Yu, USA

Abstract:

Breast cancer is the most common malignancy amongst women worldwide, accounting for approximately 2.3 million new cases and 670,000 deaths in 2022 (11). However, traditional chemotherapy treatment causes severe systemic toxicity and side effects. A new method of non-invasive drug delivery, microspheres, has been on the rise, but a clinical alternative of optimized efficacy has yet to be defined. This study proposes to synthesize polycaprolactone (PCL) microspheres encapsulating a pertuzumab and trastuzumab combination (PHESGO) to facilitate non-invasive sustained and localized release of the anti-HER2 antibodies to improve tumor reduction efficacy while minimizing toxicity of treatment.

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Introduction:

The devastation caused by breast cancer only continues to magnify, with a projected mortality toll of 42,170 women in the United States in 2025 (16). Human epidermal growth factor receptor 2 (HER2) positive breast cancer is especially invasive, in which the overproduction of HER2 receptors causes a significant acceleration in the aggressiveness of tumor growth and spread compared to other breast cancer types. Further, HER2-positive tumors historically carry poor prognosis (4). Although traditional treatments (ex: chemotherapy) have improved survival rates, many are exceedingly toxic and non-selective. In fact, 93% of breast cancer patients report treatment-related side effects (ex: severe nausea, fatigue, and pain) (7). Thus, it is increasingly imperative that an alternate method of treatment is defined. PCL polymeric microspheres, which can be injected near tumors and slowly release drug cargo over months (15)(1), are promising. The microspheres will erode away due to hydrolysis and the drug cargo will diffuse out of the particles as per Fick’s Law (13). In accordance with the Enhanced Permeability and Retention Effect, the microspheres will pass through abnormally large gaps between the endothelial cells lining blood vessels opened by the formation of a cancerous tumor, and accumulate there due to poor lymphatic drainage (3). This concentrates drug cargo around the tumor site, reducing systemic distribution and the side effects of traditional administration methods.

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Literature Review:

Research on biodegradable polymeric microspheres have shown general microsphere efficacy in cancer applications. In a notable study by Bhangoo et al. 2015, Yttrium-90 radioembolization, in which β-emitting microspheres were delivered via the hepatic artery, resulted in substantial therapeutic benefit. After Yttrium-90 microsphere administration, patients with unresectable hepatocellular carcinoma achieved partial responses or stable disease in about 48% of cases and had a median survival of about 8.4 months (5). Further, the choice of polymer carrier is critical. The common poly(lactic‐co‐glycolic) acid (PLGA) degrades relatively quickly into lactic and glycolic acids, creating an acidic microenvironment that can accelerate polymer breakdown and chemically modify or denature protein drugs (12). However, according to a study by Snehalatha et al. 2008, PCL degrades very slowly in comparison and generates minimal acidity, better preserving encapsulated cargo (17). PCL microspheres also demonstrated up to 60-day in vitro release of encapsulated chemotherapy drugs (1). With a slow and sustained release rate, microspheres can release high potency doses while sparing healthy tissues, which aligns with the therapeutic requirements of cancer treatment. Finally, anti-HER2 monoclonal antibodies are effective in tumor inhibition, but have administration flaws. Trastuzumab can significantly improve survival rates, and a dual blockade with trastuzumab and pertuzumab (PHESGO) can further improve treatment (10). However, direct delivery of PHESGO requires frequent intravenous dosing and causes accompanying systemic toxicities (ex: cardiomyopathy or heart failure) (9). This project aims to build off of the precedent of microsphere efficacy in cancer treatment to synthesize PCL microspheres with slowed release rates to deliver PHESGO, reaping the benefits of the drugs while negating the side effects that accompany direct invasive injection.

Methodology:​ 

1. Microsphere fabrication: PCL microspheres with encapsulated PHESGO will be synthesized using a double-emulsion solvent evaporation technique. An aqueous PHESGO solution containing pertuzumab, trastuzumab, and hyaluronidase will be emulsified in an organic phase of PCL dissolved in dichloromethane to form a water-in-oil emulsion. This will be emulsified again in a PVA aqueous bath under high-shear mixing. The solvent will then be allowed to evaporate to harden the spheres (14)(2). Parameters (ex: PCL concentration, PHESGO dose, mixing time) will be optimized for size and encapsulation.

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2. In vitro release kinetics: Microspheres will be incubated at 37°C in a phosphate-buffered saline with 0.1% BSA to stabilize proteins. Weekly, supernatants will be sampled and replaced. ELISA assays, which are specific to PHESGO, will be used to quantify drug release. It is expected that there will be sustained release profiles, which are characteristic of PCL. Using Korsmeyer–Peppas data to kinetic models, mechanisms of diffusion and erosion will be observed. pH levels will also be observed, which should remain neutral (characteristic of PCL), unlike PLGA microspheres which cause an acidic drop (8).

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3. In vitro bioactivity and cytotoxicity: It must be tested whether released antibodies remain active and can inhibit HER2 breast cancer cells. A HER2-overexpressing breast cancer cell line (ex: SK-BR-3) and a HER2-low control line will be used. There will be four treatment groups applied to the cell lines. (i) Free PHESGO, (ii) PHESGO-loaded microspheres, (iii) blank microspheres, (iv) no treatment. An MTT assay will be performed to measure cell viability (15). It is expected that PHESGO-loaded microspheres will reduce the viability of HER2 cells in comparison to free PHESGO, but with a slower release. The blank particle and untreated groups should have no effect. Western blotting will be performed to measure HER2 inhibition. After treating cells with PHESGO, cell lysates will be probed for phosphorylated HER2 and downstream effectors (ex: AKT, ERK) versus total protein. If HER2 inhibition was effective (ex: reduced p-AKT), that means there was also functional antibody release (8).

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4. In vivo therapeutic efficacy: Immunodeficient mice will be injected subcutaneously with SK-BR-3 cells. Once tumors reach an appropriate volume, they will be randomized and treated in four groups. (i) Free PHESGO, (ii) PHESGO-loaded microspheres, (iii) blank microspheres, (iv) no treatment. After about 10 weeks, tumors and major organs will be harvested. Using them, tumor weight and histology, HER2 pathway in tumor tissue, and any metastases will be investigated. It is anticipated that there will be slower tumor growth or regression in the PHESGO-loaded microsphere group versus the other treatment groups. Also, their blood will be tested for antibody levels and toxicity. There should be a higher tumor-to-serum ratio of antibodies, reduced peak blood levels, and lower systemic toxicity of the PHESGO-loaded microsphere group. Survival data will also be analyzed (6).

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5. Statistical analysis: A one-way ANOVA will be performed to assess the difference in cell viability, HER2 or PHESGO signal, phosphorylation levels, and tumor suppression between the treatment groups. A p-value less than 0.05 means the results are significant.

Conclusion:

This study encapsulating PHESGO in PCL microspheres has the potential to revolutionize drug delivery systems, offering alternative breast cancer therapy that is not only more effective, but has reduced systemic side effects. This would build a foundation for future studies to then replicate and apply these polymeric microspheres to interdisciplinary diseases worldwide.

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