Category: Science and technology

Water-soluble and amphiphilic phospholipid
copolymers having 2-methacryloyloxyethyl
phosphorylcholine units for the solubilization of
bioactive compounds

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Lemay We summarize the development and evaluation of new type of phospholipid polymers as a solubilizer for poorly water-soluble compounds. That is, a water-soluble and amphiphilic poly(2-methacryloyloxyethyl phosphorylcholine-random-n-butyl methacrylate) contains 30 mol% hydrophilic 2-methacryloyloxyethyl phosphorylcholine units and its weight-averaged molecular weight is around 5.0 × 104 .When the polymer is dissolved in an aqueous medium, a large portion of hydrophobic n-butyl methacrylate units assemble, forming polymer aggregates. To avoid severe biological reactions caused by conventional solubilizers, the phospholipid polymer can be applied for the solubilization of poorly water-soluble bioactive compounds. The polarity inside these polymer aggregate is the same as that of ethanol and n-butanol. Therefore, bioactive compounds, whose solubility is poor in water but good in these alcohols, can be entrapped in the polymer aggregate. The phospholipid polymer can penetrate the cell membrane by molecular diffusion, carrying inside the cell the bioactive compound, without exhibiting significant cytotoxicity. Several animal experiments have revealed that the pharmacological performance of various bioactive compound/phospholipid polymer complexes is excellent. Furthermore,functionalization of the polymer aggregate with biomolecules, such as antibodies and oligonucleotides, can be done, leading to selective capturing of the target molecules. These examples clearly indicate that water-soluble and amphiphilic phospholipid polymer is a candidate for preparing safer formulations and more effective pharmaceutical treatment with several bioactive compounds.

Poly(MPC-random-BMA) (PMB) is a typical water-soluble and amphiphilic MPC copolymer, whose weight-averaged molecular weight (Mw) is less than 5.0 × 104, and which is composed of 30 mol% MPC units in the polymer. The fact that 30 mol% MPC units in the polymer is enough to result in high water solubility of the relatively low Mw PMB,which contains hydrophobic BMA units, is a remarkable property of the MPC unit, since other hydrophilic methacrylate units, including carboxylate, sulfonate, and trimethylammonium groups, cannot provide enough solubility in water when the resulting polymers contain 70 mol% BMA units.

PMB is one of the candidate solubilizers for poorly water-soluble bioactive compounds administered orally or through the bloodstream. Water-soluble PMB of two different MWs is commercially available worldwide as PUREBRIGHT® from NOF Co. Ltd,Tokyo, Japan, as a solubilizing test kit for bioactive compounds. Moreover, many animal studies have revealed that PMB has a good potential to be used as a new solubilizer. We hope that the application of water-soluble and amphiphilic PMB will realize safer and more effective pharmaceutical treatment in the future. (Journal of Biomaterials Science, Polymer Edition, DOI: 10.1080/09205063.2017.1377023)

Strategies for Targeted Delivery of Exosomes to the Brain: Advantages and Challenges

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Delivering therapeutics to the central nervous system (CNS) is difficult because of the blood–brain barrier (BBB). Therapeutic delivery across the tight junctions of the BBB can be achieved through various endogenous transportation mechanisms. Receptor-mediated transcytosis (RMT) is one of the most widely investigated and used methods. Drugs can hijack RMT by expressing specific ligands that bind to receptors mediating transcytosis, such as the transferrin receptor (TfR), low-density lipoprotein receptor (LDLR), and insulin receptor (INSR). Cell-penetrating peptides and viral components originating from neurotropic viruses can also be utilized for the efficient BBB crossing of therapeutics. Exosomes, or small extracellular vesicles, have gained attention as natural nanoparticles for treating CNS diseases, owing to their potential for natural BBB crossing and broad surface engineering capability. RMT-mediated transport of exosomes expressing ligands such as LDLR-targeting apolipoprotein B has shown promising results. Although surface-modified exosomes possessing brain targetability have shown enhanced CNS delivery in preclinical studies, the successful development of clinically approved exosome therapeutics for CNS diseases requires the establishment of quantitative and qualitative methods for monitoring exosomal delivery to the brain parenchyma in vivo as well as elucidation of the mechanisms underlying the BBB crossing of surface-modified exosomes.

Exosomes carry various membrane proteins (e.g., CD9, CD63, PTGFRN, and Lamp2b) and lipids (e.g., phosphatidylserine) that can be utilized for the surface engineering of various targeting moieties. Engineered exosomes possessing targetability to the brain have shown promising results for CNS delivery in preclinical studies; however, they also require intense evaluation through well-designed clinical trials. For the successful development of clinically approved exosome therapeutics for CNS diseases, the establishment of imaging methods for quantitative/qualitative monitoring of exosomal delivery to the brain parenchyma in vivo and uncovering the detailed BBB crossing mechanisms of exosomes is needed. (Pharmaceutics  Daoukro 202214(3), 672)

Blockade of novel immune checkpoints and new therapeutic combinations to boost antitumor immunity

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Immunotherapy has emerged as a promising strategy for boosting antitumoral immunity. Blockade of immune checkpoints (ICs), which regulate the activity of cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells has proven clinical benefits. Antibodies targeting CTLA-4, PD-1, and PD-L1 are IC-blockade drugs approved for the treatment of various solid and hematological malignancies. However, a large subset of patients does not respond to current anti-IC immunotherapy. An integrative understanding of tumor-immune infiltrate, and IC expression and function in immune cell populations is fundamental to the design of effective therapies. The simultaneous blockade of newly identified ICs, as well as of previously described ICs, could improve antitumor response. We review the potential for novel combinatory blockade strategies as antitumoral therapy, and their effects on immune cells expressing the targeted ICs. Preclinical evidence and clinical trials involving the blockade of the various ICs are reported. We finally discuss the rationale of IC co-blockade strategy with respect to its downstream signaling in order to improve effective antitumoral immunity and prevent an increased risk of immune-related adverse events (irAEs).

Tumor growth involves a complex interplay between tumor, immune, and stromal cells, and extracellular matrix components. In the last decade, the relevance of the tumor-immune microenvironment and its direct impact on patients’ clinical outcome has become widely acknowledged. The host immune system is primed to identify and kill malignantly transformed cells to prevent tumorigenesis and tumor growth. Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells are immune cell populations responsible for immunosurveillance and, when required, for eliminating target cells. Tumor cells can be identified by CTLs as altered cells by the expression of neoantigens displayed by the major histocompatibility complex (MHC). Tumor cells expressing low levels of MHC molecules can become invisible to T cells and may escape T-cell immune control. In these cases, NK cells can identify and target cancer cells that lack MHC expression. However, tumor immune evasion, defined as the ability of tumor cells to evade the host’s immune response, happens during tumorigenesis and tumor growth. Multiple activating and inhibiting mechanisms tightly regulate the effector function of CTLs and NK cells to prevent autoimmune events and preserve tissue homeostasis. In this regard, immune checkpoints (ICs) are signaling pathways that modulate the immune response. CTLs and NK cells can express IC receptors that, when interacting with IC ligands, activate IC signaling pathways, blocking their cytotoxic activity. These IC ligands can be expressed by immunosuppressive cells, including M2-like macrophages, myeloid-derived suppressor cells (MDSCs), and T-regulatory (Treg) cells, as well as cancer cells. The continuous interaction between IC ligands and their respective IC receptors expressed by CTLs and NK, help produce a dysfunctional state in these immune cells known as exhaustion. Tumors avoid antitumoral immunity by upregulating the expression of IC ligands and recruiting immunosuppressive cells, which give rise to an immunosuppressive tumor microenvironment (TME). Tumors with a strong immunosuppressive TME have been associated with impaired immune cytotoxicity, are more aggressive, and have a poor prognosis.

Effector cells: cytotoxic T lymphocytes and natural killer cells
CTLs and NK cells are the two major immune populations that are able to eliminate malignant cells. CTLs participate in the adaptive immune response while NK cells are part of the innate immune system. Cytotoxicity arises by two pathways: Perforin/Granzyme B/Granulysin-related lysis, and death receptor-induced apoptosis. Although CTLs and NK cells act in a mechanistically similar fashion, the regulation of the activity of these immune cells, and the recognition of the targets differ. CTL cytotoxicity is acquired after T cell activation upon antigen presentation by antigen presenting cells (APCs) —mainly dendritic cells (DCs) — whereas NK cells lyse target cells without antigen presentation. When activated, CTLs and NK cells both secrete interferon (IFN)-γ and tumor necrosis factor (TNF)-α, which stimulate a pro-inflammatory immune response. Antitumoral effects have been extensively ascribed to these two immune cell populations, highlighting the relevance of comprehensively understanding the activation and inhibition mechanisms that regulate their cytotoxic activity against cancer cells by pharmacological strategies.

CTLs are defined as activated effector CD3+ CD8+ T lymphocytes and recognize target cells via the interaction between polyclonally rearranged T-cell receptor (TCR) with a peptide/MHC class I complex. Naïve CD8+ T cells interact with APCs and, upon the correct presentation of the peptide-MHC class I complex, TCR signaling causes the formation of a stabilization complex between T cells and the APC. To become fully activated, the co-stimulatory receptor CD28 must interact with its ligands, CD80 (B7.1) and CD86 (B7.2). The activity of T cells is determined by the balance of positive and negative signals from co-activator and co-inhibitory receptors when they recognize their target. To eliminate target cells, CTLs produce a stabilization complex, after which, lytic granules are secreted. Perforin forms pores in the extracellular membrane of the target cells, allowing Granzyme B and Granulysin to enter the cytosol and induce apoptosis, while membrane-bound FasL binds to its receptor Fas, inducing apoptosis in an independent manner.

Human NK cells are phenotypically defined as CD3− CD56+ lymphocytes. Two functionally distinct subsets of NK cells have been defined on the basis of their levels of expression of CD56 and CD16 (also known as Fcγ receptor III). NK cells with high-density expression of CD56 (CD56bright) and CD16− are mostly found in lymph nodes and have a great ability to release immune-modulating cytokines such as IFN-γ and TNF-α. On the other hand, low-density expression of CD56 (CD56dim) CD16+ NK cells mostly occurs in peripheral blood, where it presents a more cytotoxic phenotype characterized by high levels of Perforin and Granzyme B expression. Cytotoxic NK cell activity is independent of foreign antigens presented by MHC molecules. The balance between activation and inhibition signals, which NK cells sense through multiple innate receptors, allows the cells to respond to alterations such as cellular stress, cellular transformation, and malignancy. When activated, NK cells form a stabilization complex similarly to CTLs and release cytotoxic granules.

CTL and NK-cell activity is tightly controlled to preserve antigenic self-tolerance. Autoreactive T-cell clones are eliminated in the thymus by a process known as central tolerance. Also, a peripheral regulation of the cytotoxic response is essential to avoid inappropriate responses to self-antigens. The release of immunosuppressive molecules by M2-like macrophages and Treg cells plays a key role in establishing immune self-tolerance. Activated CTLs and NK cells upregulate the expression of multiple coinhibitory receptors, known as ICs receptors, which downregulate their cytotoxic activity when binding to their ligands, ensuring the precise regulation of their effector function (Fig. 1). Although self-tolerance mechanisms are tightly regulated, T-cell exhaustion occurs and is often observed in tumors and chronic infections. NK cells can present a similar exhausted phenotype that is characterized by stronger expression of coinhibitory receptors and weaker expression of activating receptors. Tumor-infiltrating lymphocytes (TILs) and tumor-infiltrating NK cells exhibit enhanced expression of IC receptors. This has boosted interest in understanding how these coinhibitory receptors function in order to therapeutically block them. The best characterized IC receptors are the cytotoxic T-lymphocyte-associated molecule 4 (CTLA-4) and the programmed cell death protein 1 (PD-1), but many other ICs play key central roles in controlling CTL and NK cell effector functions. (J Exp Clin Cancer Res. 2022; 41: 62.)