Development of vectors based on herpesvirus and poliovirus for oncolytic biotherapy/Разработка векторов на основе герпесвируса и полиовируса для онколитической биотерапии тема диссертации и автореферата по ВАК РФ 00.00.00, кандидат наук Ци Сяоли

• Ци Сяоли
• кандидат наук
• 2023

Оглавление диссертации кандидат наук Ци Сяоли

Table of Contents

Introduction

Chapter 1. Literature Review

1.1 Locally-acting agents

1.2 Drug delivery systems (DDSs)

1.3 Metal-organic frameworks (MOFs)

1.4 MOF-based DDSs for photodynamic treatment of chlamydial infections

1.4.1 Intracellular bacterial infections

1.4.2 Chlamydia trachomatis (C. trachomatis) infection and life cycle

1.4.3 Challenges of anti-C. trachomatis treatment

1.4.4 Using of MOF-based nanomedicines for treatment of intracellular infections

1.5 MOF as radionuclide carriers for low-dose rate (LDR) brachytherapy of cancer

1.5.1 Radiotherapy

1.5.2 LDR and HDR brachytherapy

1.5.3 Radionuclides for LDR-BT

1.5.4 3D tumor models for evaluation radiotherapeutic strategies

1.5.5 Materials for delivery of radionuclides for LDR brachytherapy

Chapter 2. Materials and Methods

2.1 Characterization of MOF nanoparticles

2.2 Fluorescent labeling of MIL-100(Fe) nanoparticles

2.3 Cell culture

2.4 Flow cytometry analysis of MIL-100(Fe)-TBO nanoparticle uptake kinetics by RAW264.7 macrophages

2.5 Intracellular localization of TBO-labeled nanoMOFs in RAW264.7 macrophages

2.6 Generation and characterization of MB-loaded nanoMOFs

2.7 In vitro analysis of photodynamic effect of MB-loaded nanoMOFs

2.8 Cell viability assay

2.9 Generation of scaffold-free (SF) A549 spheroids

2.10 Purification and analysis of total RNA and qPCR

2.11 Western blot

2.12 Immunohistochemistry analysis

2.13 Animal model

2.14 MOF nanoparticle penetration in 3D spheroids

2.15 In vitro cytotoxicity in 3D models

2.16 Intratumoral retention and biodistribution study

2.17 In vivo therapeutic effects of 90Y,Y-BTC MOFs

2.18 Statistical analysis

Chapter 3. Results

3.1 MIL-100(Fe)-MB nanoparticles for treatment of chlamydial infections

3.1.1 Characterization and post-synthesis modification of nanoMOFs

3.1.2 Cellular uptake and intracellular localization in RAW 264.7 macrophages

3.1.3 NanoMOF co-localization with C. trachomatis inclusions in infected macrophages

3.1.4 Intrinsic antibacterial activity of nanoMOFs against C. trachomatis

3.1.5 Release behavior and photodynamic activity of MB-loaded nanoMOFs in vitro

3.1.6 Photodynamic effect of MIL-100(Fe)/MB nanoparticles in infected RAW264.7 macrophages

3.2 MIL-100(Fe, Y, 90Y) and 90Y,Y-BTC MOF particles for brachytherapy of cancer

3.2.1 Synthesis and characterization of bimetallic MIL-100(Fe,Y) and Y-BTC MOF particles

3.2.2 Characterization of 3D tumor model of lung cancer

3.2.3 Expression of EMT markers, drug resistance enzymes, cytokines, and growth factors in SF A549 spheroids

3.2.4 Expression and distribution of ECM components in spheroids

3.2.5 In vitro MIL-100(Fe,Y) particle penetration to A549 spheroids

3.2.6 Evaluation of anticancer effects of MIL-100(Fe, Y,90Y) MOFs in 3D tumor model of lung cancer

3.2.7 Intratumoral retention and biodistribution study

3.2.8 90Y inhibits the growth of B16F1 spheroids

3.2.9 Therapeutic efficacy investigations of 90Y,Y-BTC MOFs

Chapter 4. Discussion

Conclusions

Main results and the outlook

List of Abbreviations

Acknowledgements

References

Введение диссертации (часть автореферата) на тему «Development of vectors based on herpesvirus and poliovirus for oncolytic biotherapy/Разработка векторов на основе герпесвируса и полиовируса для онколитической биотерапии»

Introduction

Locally-acting drug formulations are intended for administration to the tissue of interest in order to reduce systemic absorption and minimize adverse effects. Locally-acting drugs can be used for the treatment of multiple disorders including tissue injuries, infectious diseases, and malignant tumors.

In the context of intracellular bacterial infections, numerous nanomedicine platforms have been developed over the last 30 years to increase drug accumulation inside the infected cells [1,2]. Like bacterial cells, which enter the mammalian cells via endocytic/phagocytic route, nanoparticle carriers can also be internalized by the same way. It means that the use of nanocarriers as a drug delivery system (DDS) might be advantageous for eradication of intracellular pathogens.

The treatment of Chlamydiae infections is still challenging because of the invasion of these pathogens to the host cells that provides protection from the host immune system and antibacterial drugs. Two membrane barriers, including the plasma membrane and the endosomal/phagosomal membrane of the host cell, significantly restrict access of antibiotics to Chlamydia (C.) trachomatis [1,3-5]. The developmental cycle of the obligatory intracellular parasite C. trachomatis involves two alternating morphological forms such as the infectious elementary body (EB) and the proliferating reticulate body (RB). Upon internalization, EBs transform into metabolically active RBs, which modify a membrane of the intracellular vesicular compartment, termed "inclusion", with bacterial proteins that prevent lysosomal fusion [6]. The remodeled membrane subsequently provides migration of the inclusion towards the microtubule-organizing center (MTOC), which is in close proximity to the nutrient-rich peri-Golgi region [7]. The major problem for the treatment of C. trachomatis infection is the ability of this parasite to transform into a metabolically inactive persistent form, called "aberrant RB", upon the treatment with antibiotics (penicillin), gamma-interferon (IFNy), or essential nutrient deprivation. However, Chlamydia restarts proliferation and dissemination after elimination of stressful stimuli [8]. Therefore, development of novel antibacterial drugs and antimicrobial drug delivery platforms is crucial for circumventing the

defense mechanisms of C. trachomatis. Once C. trachomatis infects mainly mucosal epithelial tissues, it can be treated by locally-acting drug formulations.

Locally-acting drugs are also widely used for cancer treatment. Brachytherapy, a form of internal radiation therapy, is a common and successful cancer treatment, when the source of ionizing radiation is introduced to the tumor and destroys cancer cells that decreases the risk of the disease spreading or returning [9]. However, currently used bulky and non-biodegradable carriers for low-dose rate (LDR) brachytherapy have some limitations, such as anesthesia risks, high cost of bed immobilization, the necessity of hospitalization, potential thromboembolic problems, discomfort from vaginal packing and applicators during bed immobilization, and applicator displacement [10,11]. It is particularly likely that the seeds migrate into the lungs, which may result in an inappropriate dosimetry and possible morbidity elsewhere in the body, such as bloody sputum, pneumorrhagia, pneumothorax, and even late sequelae, including secondary cancer, when they migrate through the bloodstream [12-14]. Additionally, brachytherapy uses relatively large capsules and seeds (4.5mm in diameter, 0.8mm in length), coated with a non-degradable coating, which can cause several problems for patients, including discomfort, pain, seed embolism, when they remain permanently in the body, or migrate to the vasculature and circulate [15,16]. Therefore, developing small-size biodegradable radiation sources, which could be implanted directly into the tumor tissue and allow radionuclide retention, would be a promising therapeutic alternative to conventional brachytherapy.

Drug delivery systems (DDSs) have been extensively studied and developed in order to overcome the inherent limitations of many bioactive medications, including limited permeability across cell membranes, off-target accumulations, unregulated drug release, and undesirable side effects [1,17-20]. To improve efficacy of locally-acting drug formulations, novel engineered materials have been introduced to provide controlled/sustained drug release. Porous coordination polymers, also known as metal-organic frameworks (MOFs), have attracted a considerable interest as a promising DDS for biomedical applications. Metal-organic framework nanoparticles are formed by the self-assembly of metal ions and organic polydentate ligands [28,29]. DDSs based on MOFs provide high loading capacity due to their large surface area, ability to undergo surface functionalization, fast internalization kinetics, good biocompatibility, and biodegradability in body fluids in the treatment of cancer and

intracellular bacterial infections [18,21-24]. These properties make MOFs an appealing delivery platform for anticancer and antibacterial locally-acting agents [19,25,26].

Specifically, nanoMOFs exhibit a large diversity of structures, compositions often associated with a large pore size which, combined to their tunable and hybrid character, is suitable to encapsulate a large variety of therapeutic molecules with high efficiency and in most cases associated with a prolonged release profile [23,27,28]. In addition, they are biodegradable and can be used as a safe medication delivery vehicle if one relies on biocompatible compositions. Among them, the mesoporous iron (III) trimesate MOF denoted MIL-100(Fe) (MIL stands for "Materials of Institute Lavoisier") is particularly attractive. Its architecture is constructed from iron oxoclusters connected by trimesate moieties resulting in a mesoporous cubic architecture with two distinct cages, which contribute to co-encapsulate therapeutic agents for achieving synergistic effects [29]. Its synthesis at the nanoscale under green ambient pressure conditions, its good biocompatibility and biodegradability in body fluids, as well as its ability to undergo surface functionalization, make this nanoMOF an appealing delivery platform for anticancer and antibacterial agents [19,25,26,30].

In the study, we investigated the feasibility of using MOF-based nanomaterials as a carrier for locally-acting agents including photosensitizers for photodynamic eradication of Chlamydia infection, and radionuclides for internal radiotherapy of cancer.

To provide the rationale for using MIL-100(Fe) nanoparticles as a medication delivery vehicle to treat chlamydial infections, we evaluated here internalization kinetics of these nanoMOFs and their potential to reach chlamydial inclusions in infected macrophages. Furthermore, the intrinsic antimicrobial activity of these materials and the feasibility of their use for reducing the Chlamydia burden in infected cells was investigated. Finally, we produced these iron (III)-based nanoMOFs (MIL-100(Fe)) loaded with photosensitizer methylene blue (MB) for photodynamic treatment (PDT) of chlamydial infections (Figure 1). Antibacterial photodynamic effect of MIL-100(Fe) nanoMOFs with encapsulated MB was evaluated in infected macrophages in comparison with free photosensitizer.

Intracellular C. trachomatis Damaged bacteria

MIL-100(Fe)/MB nanoMOF

NanoMOF particle releasing MB

Figure 1. The scheme of using MIL-100(Fe)/MB nanoMOFs for photodynamic treatment of chlamydial infections.

We also investigated the feasibility of utilizing MOFs for incorporating therapeutic radioisotope yttrium-90 (90Y) (with a short half-life of 64.1 hours) into long-term biodegradable MIL-100 nanoparticles or Y-BTC microparticles. A successful 90Y isotope integration strategy for both MOF structures was developed. Tissue penetration and cytotoxicity of MOF particles were investigated using A549 3D lung spheroid model. In addition, the biodistribution of yttrium-88 was studied at different times after intratumoral administration of 88Y-containing MOF particles in two tumor-bearing mice models (C57BL/6 mice with LLC-1 and B16F1 tumors). The therapeutic effectiveness of intratumorally injected 90Y-BTC particles was further examined in C57BL/6 mice with B16F1 tumors. Goal: to study feasibility of using MOF-based nanomaterials as a carrier for locally-acting agents including photosensitizers for photodynamic eradication of Chlamydia infection, and radionuclides for internal radiotherapy of cancer. Objectives:

• To study cellular uptake and ability of nanoMOFs to accumulate in chlamydial inclusions;

• To investigate the feasibility of using photodynamic MOF nanoparticles for eradication of intracellular infection, caused by Chlamydia trachomatis;

• To investigate the feasibility of therapeutic radionuclide (90Y) incorporation into MOF structure;

• To study the penetration and cytotoxicity of MOF particles in 3D tumor spheroids;

• To analyze tissue biodistribution of MOF-incorporated 88Y after local (intratumoral)

injection.

• To evaluate the therapeutic effect of intratumoral injection of 90Y-BTC particles for the treatment of tumors.

Заключение диссертации по теме «Другие cпециальности», Ци Сяоли

Conclusions

In the study, we investigated the feasibility of using MOF-based nanomaterials as a carrier for locally-acting agents including photosensitizers for photodynamic eradication of Chlamydia infection, and radionuclides for internal radiotherapy of cancer.

The rationale for using MIL-100(Fe) nanoparticles as a medication delivery vehicle to treat chlamydial infections was proposed and studied. In this research we have shown that the fast internalization kinetics of MIL-100(Fe) nanoMOFs and the availability of MIL-100(Fe)-based MOF nanoparticles to co-localize with Chlamydia trachomatis in the infected RAW264.7 macrophages. Furthermore, MIL-100(Fe) nanoMOFs have inherent bactericidal characteristics against pathogens, hence potentially augmenting the therapeutic effectiveness of medications administered by MIL-100(Fe) nanoMOFs. The antibacterial capabilities of empty MOFs are most likely attributed to the iron-mediated Fenton reaction. Moreover, photosensitizer MB-loaded nanoMOFs, with a loading extent of 2 wt % and loading efficiency of 84 %, exhibit complete photodynamic inactivation of C. trachomatis growth. According to the results obtained, our research indicates that the created system exhibits promise in providing a photodynamic approach for efficiently eradicating Chlamydiae while minimizing harm to host cells. This approach may provide significant advantages in the management of cervical or conjunctival infections. Moreover, we propose that this novel methodology may possess substantial merit in fighting persistent chlamydial infections, since it avoids the dependence on the suppression of bacterial metabolism, a characteristic feature of conventional antibiotics.

On the other hand, successful incorporation of the therapeutic radionuclide (90Y) into long-term biodegradable MIL-100 nanoparticles and Y-BTC microparticles was developed. And MOF particles show limited penetration to 3D tumor spheroids that presumably hinders their diffusive transport outside tumor tissue. The establishment of therapeutic dose for the aim of studying radiotherapy in vivo was achieved by evaluating the cytotoxic impacts of different formulations using 3D spheroid models. Furthermore, experimental evidence shown that yttrium exhibited sustained retention inside tumors during in vivo biodistribution after the

administration of MOF-incorporated 88Y by local injection. Meanwhile, 90Y,Y-BTC MOFs significantly inhibited the tumor growth after intratumoral injection. The findings indicate that the developed methods, which include the incorporation of radionuclide into MOFs particles, have significant promise for the treatment of tumors. Moreover, the use of MOFs particles as a platform has the potential to be integrated with other therapeutic modalities, hence enabling the investigation of a synergistic combination treatment in future research endeavors.

Therefore, the results presented in this study collectively prove the potential of metal-organic frameworks as an appealing strategy for delivering locally active pharmaceuticals to effectively combat antibacterial infections and anticancer treatments.

Main results and the outlook

1) The study shows the fast internalization kinetics of MIL-100(Fe) nanoMOFs and the availability of MIL-100(Fe)-based MOF nanoparticles to co-localize with Chlamydia trachomatis in the infected RAW264.7 macrophages.

2) The measurement of reactive oxygen species (ROS) generation in vitro demonstrated that the incubation of the nanoMOFs with free MB after light irradiation led to a 1.5fold increase of ROS generation over time in comparison with free MB.

3) Simultaneous infection and treatment of RAW264.7 cells with empty nanoMOFs resulted in a bacterial load reduction from 100 to 36% that indicates an intrinsic anti-chlamydial effect of this iron-containing nanomaterial.

4) NanoMOFs loaded with photosensitizer methylene blue (MB) exhibit complete photodynamic inactivation of C. trachomatis growth as compared with non-irradiated control.

5) The yttrium-incorporated MOF particles both exhibit excellent stability in different solutions, with roughly 15 % yttrium release from MIL-100(Fe, Y,88Y) in FBS solution after 120 hours and less than 5 % yttrium release from Y,88Y-BTC MOFs in 6 days at 37 0C.

6) MOF particles show limited penetration to 3D lung tumor spheroids that presumably hinders their diffusive transport outside tumor tissue.

7) By evaluating the cytotoxicity of bimetallic MOF nanoparticles in the A549 3D lung spheroid model, the therapeutic dose for further in vivo biodistribution investigation was established.

8) In vivo biodistribution, the therapeutic payload of MOF particles with 88Y incorporation were mainly maintained inside the tumors with negligible leakage to other organs, which would not only improve treatment effectiveness but also reduce unfavorable toxic effects associated with radiation damage to normal organs.

9) The therapeutic dosage of yttrium-90 for the radiotherapy experiment was determined from an examination of the cytotoxic effects of free yttrium at different doses on the

B16F1 cell spheroids.

10) In vivo results obtained from treating melanoma-bearing mice showed that 90Y-BTC MOF particles remarkably prolonged survival.