Peritoneal B Cells Play a Role in the Production of Anti-polyethylene Glycol (PEG) IgM against Intravenously Injected siRNA-PEGylated Liposome Complexes.

Polyethylene glycol (PEG)-modified (PEGylated) cationic liposomes are frequently used as delivery vehicles for small interfering RNA (siRNA)-based drugs because of their ability to encapsulate/complex with siRNA and prolong the circulation half-life in vivo. Nevertheless, we have reported that subsequent intravenous (IV) injections of siRNA complexed with PEGylated cationic liposomes (PLpx) induces the production of anti-PEG immunoglobulin M (IgM), which accelerates the blood clearance of subsequent doses of PLpx and other PEGylated products. In this study, it is interesting that splenectomy (removal of spleen) did not prevent anti-PEG IgM induction by IV injection of PLpx. This indicates that B cells other than the splenic version are involved in anti-PEG IgM production under these conditions. In vitro and in vivo studies have shown that peritoneal cells also secrete anti-PEG IgM in response to the administration of PLpx. Interleukin-6 (IL-6) is a glycoprotein that is secreted by peritoneal immune cells and has been detected in response to the in vivo administration of PLpx. These observations indicate that IV injection of PLpx stimulates the proliferation/differentiation of peritoneal PEG-specific B cells into plasma cells via IL-6 induction, which results in the production of anti-PEG IgM from the peritoneal cavity of mice. Our results suggest the mutual contribution of peritoneal B cells as a potent anti-PEG immune response against PLpx.

[Characterization of Supersulfide in Serum Albumin and Its Therapeutic Application].

Recent studies have shown that proteins already possess supersulfides during the translation. However, the distribution and the role of supersulfides are not fully understood. In this review, we focus on supersulfides in biological fluids, especially in serum. Various methods for measuring supersulfides have been developed, and these methods have elucidated the presence of supersulfides in serum proteins including serum albumin. Since the levels of supersulfides in serum and serum albumin of patients with chronic kidney disease were lower than those in healthy subjects and recovered by hemodialysis, the levels of supersulfides in serum would be an indicator reflecting oxidative stress. In addition, it has long been known that serum albumin is responsible for sulfur transference. We have applied this phenomenon to the synthesis of sulfur-added albumin (Sn-HSA) by the reaction of serum albumin with sodium polysulfide (Na2Sn). Sn-HSA suppressed the melanin production via scavenging oxidative stress. As described above, studies on the characterization of supersulfides in serum albumin may contribute to the monitoring of redox balance and prevention of oxidative stress-related diseases.

Absence of Anaphylactic Reactions to Injection of Hemoglobin Vesicles (Artificial Red Cells) to Rodents.

The safety and efficacy of hemoglobin vesicles (HbVs) as artificial oxygen carriers encapsulating a purified and concentrated Hb solution in liposomes have been studied extensively. The HbV surface, modified with PEG by incorporating a PEG-conjugated phospholipid, is beneficial for storage and biocompatibility. However, it might be possible that interaction of PEG and the pre-existing anti-PEG antibody in the bloodstream causes acute adverse reaction. This study used two sets of experiments with rats and guinea pigs to ascertain whether the anti-PEG antibody generated by the PEG-modified HbV injection can induce anaphylactic reactions. SD rats received repeated intravenous injection of HbV at a dose rate of 16 or 32 mL/kg three times. Not anti-PEG IgG but anti-PEG IgM was detected. Nevertheless, no anaphylactic reaction occurred. Guinea pigs were used to study the presence of active systemic anaphylaxis further after injections of the PEG-modified liposomes used for HbV. The animals were sensitized by three repeated subcutaneous injections of PEG-modified liposomes (PEG-liposome) along with adjuvant at 1 week intervals. For comparison, unmodified liposomes (liposome) and 10 times excessively PEG-modified liposomes with ionizable lipid (10PEG-DODAP-liposome) were used. Inclusion of PEG modification induced not only anti-PEG IgM but also anti-PEG IgG. Three weeks after the final injection, intravenous injection of both PEG-liposome and liposome (1 mL/kg) induced no anaphylactic reaction. However, the injection of 10PEG-DODAP-liposome showed one lethal anaphylaxis case and one mild anaphylaxis case. Antisera obtained from the animal sensitized as described above were inoculated (0.05 mL) intradermally into fresh guinea pigs. The presence of passive cutaneous anaphylaxis was evaluated after intravenous injections (1 mL/kg) of three liposomes with Evans blue. No dye leakage was detected at any inoculated skin point for PEG-liposome or liposome, but a slight leakage was detected in one inoculated skin point for 10PEG-DODAP-liposome. These results indicate the absence of acute allergic reactions at repeated injections of HbVs despite the anti-PEG antibody induction. Not all the PEG-modified liposomes show anaphylaxis, and it may depend on the amount of PEGylated phospholipid and lipid composition of PEG-modified liposomes.

Acute Kidney Injury Caused by Rhabdomyolysis Is Ameliorated by Serum Albumin-Based Supersulfide Donors through Antioxidative Pathways.

Oxidative stress is responsible for the onset and progression of various kinds of diseases including rhabdomyolysis-induced acute kidney injury (AKI). Antioxidants are, therefore, thought to aid in the recovery of illnesses linked to oxidative stress. Supersulfide species have been shown to have substantial antioxidative activity; however, due to their limited bioavailability, few supersulfide donors have had their actions evaluated in vivo. In this study, human serum albumin (HSA) and N-acetyl-L-cysteine polysulfides (NACSn), which have polysulfides in an oxidized form, were conjugated to create a supersulfide donor. HSA is chosen to be a carrier of NACSn because of its extended blood circulation and high level of biocompatibility. In contrast to a supersulfide donor containing reduced polysulfide in HSA, the NACSn-conjugated HSAs exhibited stronger antioxidant activity than HSA and free NACSn without being uptaken by the cells in vitro. The supersulfide donor reduced the levels of blood urea nitrogen and serum creatinine significantly in a mouse model of rhabdomyolysis-induced AKI. Supersulfide donors significantly reduced the expression of oxidative stress markers in the kidney. These results indicate that the developed supersulfide donor has the therapeutic effect on rhabdomyolysis-induced AKI.

Impact of Anti-PEG IgM Induced via the Topical Application of a Cosmetic Product Containing PEG Derivatives on the Antitumor Effects of PEGylated Liposomal Antitumor Drug Formulations in Mice.

Poly(ethylene glycol) (PEG) is used in many common products, such as cosmetics. PEG, however, is also used to covalently conjugate drug molecules, proteins, or nanocarriers, which is termed PEGylation, to serve as a shield against the natural immune system of the human body. Repeated administration of some PEGylated products, however, is known to induce anti-PEG antibodies. In addition, preexisting anti-PEG antibodies are now being detected in healthy individuals who have never received PEGylated therapeutics. Both treatment-induced and preexisting anti-PEG antibodies alter the pharmacokinetic properties, which can result in a subsequent reduction in the therapeutic efficacy of administered PEGylated therapeutics through the so-called accelerated blood clearance (ABC) phenomenon. Moreover, these anti-PEG antibodies are widely reported to be related to severe hypersensitivity reactions following the administration of PEGylated therapeutics, including COVID-19 vaccines. We recently reported that the topical application of a cosmetic product containing PEG derivatives induced anti-PEG immunoglobulin M (IgM) in a mouse model. Our finding indicates that the PEG derivatives in cosmetic products could be a major cause of the preexistence of anti-PEG antibodies in healthy individuals. In this study, therefore, the pharmacokinetics and therapeutic effects of Doxil (doxorubicin hydrochloride-loaded PEGylated liposomes) and oxaliplatin-loaded PEGylated liposomes (Liposomal l-OHP) were studied in mice. The anti-PEG IgM antibodies induced by the topical application of cosmetic products obviously accelerated the blood clearance of both PEGylated liposomal formulations. Moreover, in C26 tumor-bearing mice, the tumor growth suppressive effects of both Doxil and Liposomal l-OHP were significantly attenuated in the presence of anti-PEG IgM antibodies induced by the topical application of cosmetic products. These results confirm that the topical application of a cosmetic product containing PEG derivatives could produce preexisting anti-PEG antibodies that then affect the therapeutic efficacy of subsequent doses of PEGylated therapeutics.

Treatment-induced and Pre-existing Anti-peg Antibodies: Prevalence, Clinical Implications, and Future Perspectives.

Polyethylene glycol (PEG) is a versatile polymer that is used in numerous pharmaceutical applications like the food industry, a wide range of disinfectants, cosmetics, and many commonly used household products. PEGylation is the term used to describe the covalent attachment of PEG molecules to nanocarriers, proteins and peptides, and it is used to prolong the circulation half-life of the PEGylated products. Consequently, PEGylation improves the efficacy of PEGylated therapeutics. However, after four decades of research and more than two decades of clinical applications, an unappealing side of PEGylation has emerged. PEG immunogenicity and antigenicity are remarkable challenges that confound the widespread clinical application of PEGylated therapeutics - even those under clinical trials - as anti-PEG antibodies (Abs) are commonly reported following the systemic administration of PEGylated therapeutics. Furthermore, pre-existing anti-PEG Abs have also been reported in healthy individuals who have never been treated with PEGylated therapeutics. The circulating anti-PEG Abs, both treatment-induced and pre-existing, selectively bind to PEG molecules of the administered PEGylated therapeutics inducing activation of the complement system, which results in remarkable clinical implications with varying severity. These include increased blood clearance of the administered PEGylated therapeutics through what is known as the accelerated blood clearance (ABC) phenomenon and initiation of serious adverse effects through complement activation-related pseudoallergic reactions (CARPA). Therefore, the US FDA industry guidelines have recommended the screening of anti-PEG Abs, in addition to Abs against PEGylated proteins, in the clinical trials of PEGylated protein therapeutics. In addition, strategies revoking the immunogenic response against PEGylated therapeutics without compromising their therapeutic efficacy are important for the further development of advanced PEGylated therapeutics and drug-delivery systems.

Encapsulation of an Antioxidant in Redox-Sensitive Self-Assembled Albumin Nanoparticles for the Treatment of Hepatitis.

Hepatitis is an inflammation of the liver caused by the inadequate elimination of reactive oxygen species (ROS) derived from Kupffer cells. Edaravone is clinically used as an antioxidant but shows poor liver distribution. Herein, we report on the design of a Kupffer cell-oriented nanoantioxidant based on a disulfide cross-linked albumin nanoparticle containing encapsulated edaravone (EeNA) as a therapeutic for the treatment of hepatitis. Since the edaravone is bound to albumin, this results in a soluble and stable form of edaravone in water. Exchanging the intramolecular disulfide bonds to intermolecular disulfide bridges of albumin molecules allowed the preparation of a redox responsive albumin nanoparticle that is stable in the blood circulation but can release drugs into cells. Consequently, EeNA was fabricated by the nanoscale self-assembly of edaravone and albumin nanoparticles without the additives that are contained in commercially available edaravone preparations. EeNA retained its nanostructure under serum conditions, but the encapsulated edaravone was released efficiently under intracellular reducing conditions in macrophages. The EeNA was largely distributed in the liver and subsequently internalized into Kupffer cells within 60 min after injection in a concanavalin-A-induced hepatitis mouse. The survival rate of the hepatitis mice was significantly improved by EeNA due to the suppression of liver necrosis and oxidative stress by scavenging excessive ROS. Moreover, even through the postadministration, EeNA showed an excellent hepatoprotective action as well. In conclusion, EeNA has the potential for use as a nanotherapeutic against various types of hepatitis because of its Kupffer cell targeting ability and redox characteristics.

Anti-PEG IgM production induced by PEGylated liposomes as a function of administration route.

Modifying the surface of nanoparticles with polyethylene glycol (PEG) is a commonly used approach for improving the in vitro stability of nanoparticles such as liposomes and increasing their circulation half-lives. We have demonstrated that, in certain conditions, an intravenous (i.v.) injection of PEGylated liposomes (PEG-Lip) induced anti-PEG IgM antibodies, which led to rapid clearance of second doses in mice. SARS-CoV-2 vaccines, composed of mRNA-containing PEGylated lipid nanoparticles, have been widely administered as intramuscular (i.m.) injections, so it is important to determine if PEGylated formulations can induce anti-PEG antibodies. If the favorable properties that PEGylation imparts to therapeutic nanoparticles are to be widely applicable this should apply to various routes of administration. However, there are few reports on the effect of different administration routes on the in vivo production of anti-PEG IgM. In this study, we investigated anti-PEG IgM production in mice following i.m., intraperitoneal (i.p.) and subcutaneous (s.c.) administration of PEG-Lip. PEG-Lip appeared to induce anti-PEG IgM by all the tested routes of administration, although the lipid dose causing maximum responses varied. Splenectomy attenuated the anti-PEG IgM production for all routes of administration, suggesting that splenic immune cells may have contributed to anti-PEG IgM production. Interestingly, in vitro experiments indicated that not only splenic cells but also cells in the peritoneal cavity induced anti-PEG IgM following incubation with PEG-Lip. These observations confirm previous experiments that have shown that measurable amounts of PEG-Lip administered i.p., i.m. or s.c. are absorbed to some extent into the blood circulation, where they can be distributed to the spleen and/or peritoneal cavity, and are recognized by B cells, triggering anti-PEG IgM production. The results obtained in this study have important implications for developing efficient PEGylated nanoparticular delivery system.

A polyethylene glycol-conjugate of deoxycytidine analog, DFP-14927, produces potential antitumor effects on pancreatic tumor-xenograft murine models via inducing G2/M arrest.

A deoxycytidine analog is a potential agent for the treatment of several cancers, which includes poorly prognostic pancreatic cancer. We previously developed deoxycytidine analog DFP-10917, and long-term/low-dose infusions of this analog has produced antitumor effects in leukemia cancer- and ovarian cancer-xenograft models. DFP-10917 is now undergoing clinical Phase III study in the United States for the treatment of patients with relapsed or refractory acute myeloid leukemia. PEG-drug conjugation has become a promising technique to improve the pharmacokinetic and pharmacodynamic properties of anti-cancer drugs. In the present study, we synthesized a novel PEG-drug conjugate of DFP-10917, referred to hereafter as DFP-14927, using a 4-armed CTPEG system to endow the DFP-10917 drug with favorable long-circulating properties that maximize its utility and antitumor efficacy. Intravenous injection of the synthesized DFP-14927 returned encouraging antitumor effects in a Panc-1 human pancreatic tumor- and a BxPC-3 human pancreatic tumor-xenograft models. These effects were comparable to that of free DFP-10917 as well as to that of gemcitabine, which is considered a standard in the treatment of pancreatic cancer. In vitro studies revealed that DFP-14927 inhibits cell division on human pancreatic cancer cell lines via arrest of the G2/M phase in the cell cycle, which is consistent with the effects of free DFP-10917. Intravenous administration of the newly synthesized DFP-14927 has induced G2/M arrest in human pancreatic tumor-xenograft murine models, which represents an improvement in the pharmacokinetics of DFP-10917. DFP-14927 could be an alternative for patients who cannot accept prolonged or continuous infusions of DFP-10917.

The Post-insertion Method for the Preparation of PEGylated Liposomes.

PEGylation is a crucial process for decorating the surface of liposomes with polyethylene glycol (PEG) for clinical use. This process endows the liposomes extended circulation time and improved stability in vivo. The post-insertion method is one of the well-established techniques for PEGylation. This method requires only one-step incubation to accomplish the transfer of PEGylated lipids from PEGylated lipid-based micelles into the membranes of preformed liposomes.

Potential Efficacy of Proteasome Inhibitor, Delanzomib, for the Treatment of Renal Fibrosis.

Renal fibrosis is scarring and tissue hardening caused by the excess deposition of extracellular matrix proteins in response to chronic inflammation. Renal fibrosis is the primary cause of a progressive loss of renal function, and is an important therapeutic target because it ultimately leads to end-stage renal failure, which can be treated only by either dialysis or kidney transplantation. There is no effective treatment that specifically targets renal fibrosis. Myofibroblasts are known to evade apoptosis by activating molecular mechanisms in response to pro-survival biomechanical and growth factor signals from the fibrotic microenvironment. In this study, we screened and selected compounds that selectively cause cell death in myofibroblasts in vitro and studied their possible potency against renal fibrosis in a mouse model. Several proteasome inhibitors induced selective cell death in myofibroblasts differentiated from the human fibroblast cell line (MRC5). The in vivo antifibrotic effect of Delanzomib (Dz), one of the proteasome inhibitors most sensitive to myofibroblasts in vitro, was investigated in a Unilateral Ureteric Obstruction (UUO) mouse model. Treatment with Dz decreased the expression levels of the actin-alpha-2 (ACTA2) and collagen-type-1-alpha-1 (COL1A1) genes in the kidney, which are common fibrosis markers. These results suggest that Dz might be a compound that suppresses renal fibrosis by inducing selective cell death of myofibroblasts, although further investigation is required.

Ionic Liquid-Based Transcutaneous Peptide Antitumor Vaccine: Therapeutic Effect in a Mouse Tumor Model.

Traditional vaccinations need to be injected with needles, and since some people have a strong aversion to needles, a needle-free alternative delivery system is important. In this study, we employed ionic liquids (ILs) for transcutaneous delivery of cancer antigen-derived peptides to obtain anticancer therapeutic effects in a needle-free manner. ILs successfully increased the in vitro skin permeability of a peptide from Wilms tumor 1 (WT1), one of the more promising cancer antigens, plus or minus an adjuvant, resiquimod (R848), a toll-like receptor 7 agonist. In vivo studies demonstrated that concomitant transcutaneous delivery of WT1 peptide and R848 by ILs induced WT1-specific cytotoxic T lymphocyte (CTL) in mice, resulting in tumor growth inhibition in Lewis lung carcinoma-bearing mice. Interestingly, administrating R848 in ILs before WT1 peptides in ILs increased tumor growth inhibition effects compared to co-administration of both. We found that the prior application of R848 increased the infiltration of leukocytes in the skin and that subsequent delivery of WT1 peptides was more likely to induce WT1-specific CTL. Furthermore, sequential immunization with IL-based formulations was applicable to different types of peptides and cancer models without induction of skin irritation. IL-based transcutaneous delivery of cancer antigen-derived peptides and adjuvants, either alone or together, could be a novel approach to needle-free cancer therapeutic vaccines.

Investigation of anti-PEG antibody response to PEG-containing cosmetic products in mice.

Polyethylene glycol (PEG), a polyether compound, is available in molecular weights from ∼300 g/mol to ∼10,000,000 g/mol. In the molecular weight range of ∼750 to ∼5000, PEG is commonly used in bioconjugation technology and nano-formulations to improve the circulation half-life of the formulations and increase their stability. In cosmetics, lower molecular weight PEG compounds such as PEG 60 or PEG 100 are widely used as emulsifiers and skin penetration enhancers. PEG polymers are generally recognized as biologically inert and non-immunogenic. However, it is recently reported that the "pre-existing" anti-PEG antibodies have been detected in high percentages of healthy individuals who have never received treatment with parenteral PEGylated formulations. To the best of our knowledge, we are the first to attempt to find an explanation for the source of pre-existing anti-PEG antibodies in healthy individuals. In a murine study, we demonstrated that topically applied PEG derivatives, present in two commercially available cosmetic products, could efficiently penetrate the stratum corneum and reach the systemic circulation. The skin penetration of PEG derivatives was further enhanced in injured or otherwise compromised skin. Daily application of cosmetic PEG derivatives primed the immune system, inducing anti-PEG IgM production. Anti-PEG IgM was detected by Day 14 in mice with normal skin, while anti-PEG IgM was detected as early as day 7 in mice with compromised skin. In addition, in mice with pre-induced circulating levels of anti-PEG IgM, topically applied PEG derivatives from cosmetic products appeared to bind to the pre-induced anti-PEG IgM, lowering blood levels. Current results indicate that PEG derivatives in cosmetic products may be an important contributor to the source of the "pre-existing" anti-PEG antibodies that have been detected in healthy individuals.

Clinical Impact of Antipolyethylene Glycol (PEG) Antibody in Hematological Patients Administered PEGylated-Granulocyte Colony-Stimulating Factor.

Polyethylene glycol (PEG) is a polymer covalently attached to proteins to improve their half-life and efficacy. We previously reported that the PEGylated granulocyte colony-stimulating factor (PEG-G-CSF) is immunogenic, which could adversely impact drug efficacy and safety in animal models. Here, we analyzed the relationship between anti-PEG antibody titers and the clinical impact of PEG-G-CSF in 19 hematological patients. A gradual decrease of anti-PEG antibody titers from baseline was observed after PEG-G-CSF administration. Of the 19 participants, 10 were assessed for noninfectious fever after the first administration of PEG-G-CSF and three experienced this reaction. The receiver operating characteristic curve revealed that the cut-off values of pretreated anti-PEG IgM and IgG titers for noninfectious fever were set at 5.0 and 96.6 U/mL, respectively. All patients who experienced noninfectious fever had anti-PEG antibody titers above this cut-off value (P = .033). An enzyme-linked immunosorbent assay revealed that some anti-PEG antibodies in patients with anti-PEG antibody titers above the cut-off value reacted with the PEGylated liposome. These results indicate the reactivity of the anti-PEG antibodies to PEGylated therapeutics observed in hematologic patients and the possibility of the relationship between high titers of anti-PEG antibodies and the development of adverse events after PEG-G-CSF administration.

FTY720 Reduces Lipid Accumulation by Upregulating ABCA1 through Liver X Receptor and Sphingosine Kinase 2 Signaling in Macrophages.

Formation of foam cells as a result of excess lipid accumulation by macrophages is a pathological hallmark of atherosclerosis. Fingolimod (FTY720) is an immunosuppressive agent used in clinical settings for the treatment of multiple sclerosis and has been reported to inhibit atherosclerotic plaque development. However, little is known about the effect of FTY720 on lipid accumulation leading to foam cell formation. In this study, we investigated the effects of FTY720 on lipid accumulation in murine macrophages. FTY720 treatment reduced lipid droplet formation and increased the expression of ATP-binding cassette transporter A1 (ABCA1) in J774 mouse macrophages. FTY720 also enhanced the expression of liver X receptor (LXR) target genes such as FASN, APOE, and ABCG1. In addition, FTY720-induced upregulation of ABCA1 was abolished by knockdown of sphingosine kinase 2 (SphK2) expression. Furthermore, we found that FTY720 treatment induced histone H3 lysine 9 (H3K9) acetylation, which was lost in SphK2-knockdown cells. Taken together, FTY720 induces ABCA1 expression through SphK2-mediated acetylation of H3K9 and suppresses lipid accumulation in macrophages, which provides novel insights into the mechanisms of action of FTY720 on atherosclerosis.

A Maleimide-Terminally Modified PEGylated Liposome Induced the Accelerated Blood Clearance Independent of the Production of Anti-PEG IgM Antibodies.

PEGylated liposomes (PL) lose their long-circulating characteristic when administered repeatedly, called the accelerated blood clearance (ABC) phenomenon. The ABC phenomenon is generally thought to occur when the anti-polyethylene glycol (PEG) antibody (anti-PEG immunoglobulin M (IgM)) expressed in the spleen B cells triggered by the first dose of PL binds to the second and subsequent doses of PL, leading to activation of the complement system. MAL-PEG-DSPE, a PEG lipid with a maleimide (MAL) group at the PEG terminal, is used in various studies as a linker for ligand-bound liposomes such as antibody-modified liposomes. However, most ABC phenomenon research used PL with a terminal methoxy group (PL-OCH3). In this study, we prepared MAL-PEG-DSPE liposomes (PL-MAL) to evaluate the effect of PL-MAL on the ABC phenomenon induction compared to PL-OCH3. Pharmacokinetic, anti-PEG IgM secretion and complement activation analyses of these liposomes were conducted in mice. Interestingly, despite C3 bound to the surface of the initially administered PL-MAL, the administered PL-MAL showed high blood retention, demonstrating the same results as PL-OCH3. On the other hand, although the secretion of anti-PEG IgM induced by PL-MAL was lower than PL-OCH3, the second dose of PL-MAL rapidly disappeared from the blood. These results suggest that the antibody produced from the first dose of PL-MAL binds to the second dose of PL-MAL, thereby activating C3 to act as an opsonin which promotes phagocytic uptake. In conclusion, PL-MAL induced the ABC phenomenon independent of the production of IgM antibodies against PEG. This study provides valuable findings for further studies using ligand-bound liposomes.

Wnt2b and Wnt5a expression is highly associated with M2 TAMs in non­small cell lung cancer.

Tumor­associated macrophages (TAMs), particularly M2 macrophages, promote tumor progression, while Wnt genes encode a family of multi­functional glycoproteins that serve an important role in tumorigenesis. Immunohistochemical studies were performed to evaluate Wnt2b and Wnt5a expression in tumor and stromal cells in M2 and M1 TAMs and Ki­67 proliferation index in 160 consecutive patients with resected non­small cell lung cancer (NSCLC). Overall, 52 tumors (32.5%) were classified as tumoral Wnt2b­high (Wnt2b­positive tumor cells >30%) and 95 (59.4%) as stromal Wnt2b­high (Wnt2b­positive stromal cells >30%), while 75 (46.9%) were classified as tumoral Wnt5a­high (Wnt5a­positive tumor cells >30%) and 63 (39.4%) as stromal Wnt5a­high (Wnt5a­positive stromal cells >28%). The density of M2 TAMs was significantly higher in the tumoral (P=0.0024) and stromal Wnt2b­high groups (P=0.0054). The density of M2 TAMs was also significantly higher in the tumoral (P=0.0005) and stromal Wnt5a­high groups (P=0.0486). By contrast, no difference in stromal or islet M1 TAM density was observed in relation to tumoral or stromal Wnt2b or Wnt5a status. Furthermore, Ki­67 proliferation index was significantly higher in the tumoral (P=0.0121) and stromal Wnt2b­high (P=0.0019) and tumoral Wnt5a­high (P=0.0088) groups. Overall survival rate was significantly lower in the Wnt2b­high (P=0.0437), Wnt5a­high (P=0.0106) and M2 TAM­high (P=0.0060) groups. Wnt2b and Wnt5a expression in tumor and stromal cells may induce M2 TAMs to produce more aggressive behavior during tumor progression in NSCLC.

Polyethylene glycol (PEG): The nature, immunogenicity, and role in the hypersensitivity of PEGylated products.

Polyethylene glycol (PEG) is a versatile polymer that is widely used as an additive in foods and cosmetics, and as a carrier in PEGylated therapeutics. Even though PEG is thought to be less immunogenic, or perhaps even non-immunogenic, with a variety of physicochemical properties, there is mounting evidence that PEG causes immunogenic responses when conjugated with other materials such as proteins and nanocarriers. Under these conditions, PEG with other materials can result in the production of anti-PEG antibodies after administration. The antibodies that are induced seem to have a deleterious impact on the therapeutic efficacy of subsequently administered PEGylated formulations. In addition, hypersensitivity to PEGylated formulations could be a significant barrier to the utility of PEGylated products. Several reports have linked the presence of anti-PEG antibodies to incidences of complement activation-related pseudoallergy (CARPA) following the administration of PEGylated formulations. The use of COVID-19 mRNA vaccines, which are composed mainly of PEGylated lipid nanoparticles (LNPs), has recently gained wide acceptance, although many cases of post-vaccination hypersensitivity have been documented. Therefore, our review focuses not only on the importance of PEGs and its great role in improving the therapeutic efficacy of various medications, but also on the hypersensitivity reactions attributed to the use of PEGylated products that include PEG-based mRNA COVID-19 vaccines.

Oral administration of sodium bicarbonate can enhance the therapeutic outcome of Doxil® via neutralizing the acidic tumor microenvironment.

The pH of the tumor microenvironment in solid tumors is reported to be more acidic than that of normal tissues. The pH is controlled by over-expression of several transporters that are associated with the progression, angiogenesis, and metastasis of solid tumors. Antitumor effects of weak-base anticancer agents, such as doxorubicin (DXR), could be reduced in an acidic environment because of increases in the ionized form of the drug under these conditions, reducing its membrane penetrability. In our previous studies, we demonstrated that oral administration of sodium bicarbonate (NaHCO3) can neutralize the acidic tumor microenvironment and enhance the effects of small molecule anticancer drugs. However, it is not known whether or not increasing the tumor pH by oral administration of NaHCO3 leads to enhanced antitumor effects of lipidic nanoparticle formulations of weak-base anticancer drugs, such as Doxil®. In this study, we investigated the antitumor efficacy of Doxil® in combination with oral administration of NaHCO3 in a Colon26 tumor-bearing mouse model. NaHCO3 clearly enhanced the tumor-growth inhibitory effect of Doxil® without exacerbating any systemic side effects. In vitro studies indicated that high levels of DXR were internalized into cells at neutral pH. These studies demonstrate that the neutralization of acidic tumor microenvironment by an oral administration of NaHCO3 could be a promising approach to enhance the therapeutic outcomes of Doxil®.