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.

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.

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.

Electrospun Nanofibers Revisited: An Update on the Emerging Applications in Nanomedicine.

Electrospinning (ES) has become a straightforward and customizable drug delivery technique for fabricating drug-loaded nanofibers (NFs) using various biodegradable and non-biodegradable polymers. One of NF's pros is to provide a controlled drug release through managing the NF structure by changing the spinneret type and nature of the used polymer. Electrospun NFs are employed as implants in several applications including, cancer therapy, microbial infections, and regenerative medicine. These implants facilitate a unique local delivery of chemotherapy because of their high loading capability, wide surface area, and cost-effectiveness. Multi-drug combination, magnetic, thermal, and gene therapies are promising strategies for improving chemotherapeutic efficiency. In addition, implants are recognized as an effective antimicrobial drug delivery system overriding drawbacks of traditional antibiotic administration routes such as their bioavailability and dosage levels. Recently, a sophisticated strategy has emerged for wound healing by producing biomimetic nanofibrous materials with clinically relevant properties and desirable loading capability with regenerative agents. Electrospun NFs have proposed unique solutions, including pelvic organ prolapse treatment, viable alternatives to surgical operations, and dental tissue regeneration. Conventional ES setups include difficult-assembled mega-sized equipment producing bulky matrices with inadequate stability and storage. Lately, there has become an increasing need for portable ES devices using completely available off-shelf materials to yield highly-efficient NFs for dressing wounds and rapid hemostasis. This review covers recent updates on electrospun NFs in nanomedicine applications. ES of biopolymers and drugs is discussed regarding their current scope and future outlook.

A mouse model for studying the effect of blood anti-PEG IgMs levels on the in vivo fate of PEGylated liposomes.

The presence of anti-polyethylene glycol (PEG) antibodies in the systemic circulation might have potential implications for the therapeutic activity of PEGylated products in vivo in the clinic. In order to study the effect of pre-existing anti-PEG antibodies on the in vivo fate and the therapeutic efficiency of PEGylated therapeutics, we developed a BALB/c mouse model by virtue of the intraperitoneal (i.p.) inoculation of hybridoma cells (HIK-M09 and HIK-M11), secreting monoclonal anti-PEG IgM, mimicking the presence of pre-existing anti-PEG antibodies in the blood. In the model, the titers of anti-PEG IgM in the blood increased as a function of hybridoma cells numbers and time after i.p. inoculation. The in vivo levels of anti-PEG IgM decreased in a dose-dependent manner, following i.v. administration of empty PEGylated liposomes. C26 tumor-bearing mice with measurable levels of anti-PEG IgM, receiving i.v. injection of DiR-labeled empty PEGylated liposomes, showed lower levels of liposomal tumor accumulation and higher levels of liver and spleen accumulation, compared to C26 tumor-bearing mice without measurable anti-PEG IgM. This specifies that the presence of anti-PEG IgM in the murine circulation induced accelerated blood clearance of PEGylated liposomes and reduced their tumor accumulation. The biodistribution and antitumor efficacy of commercially available doxorubicin (DXR)-containing PEGylated liposomes, Doxil®, were scrutinized in the anti-PEG IgM mouse model. In C26 tumor-bearing mice having circulating anti-PEG IgM, at 24 h after injection almost no DXR was observed in blood and tumor, and increased DXR accumulation was observed in spleen and liver, compared to tumor-bearing mice with no circulating anti-PEG IgM. The antitumor efficacy of Doxil® was significantly compromised in the C26 tumor-bearing mice in the presence of anti-PEG IgM. These results demonstrate that the anti-PEG IgM mouse model could be a useful prognostic indicator for the therapeutic effectiveness of different formulations of PEGylated therapeutics in pre-clinical studies.

Using Bio-Layer Interferometry to Evaluate Anti-PEG Antibody-Mediated Complement Activation.

The purpose of this study was to develop a Bio-layer interferometry (BLI) system that could be an alternative approach for the direct evaluation of anti-polyethylene glycol (PEG) immunoglobulin M (IgM)-mediated complement activation of the accelerated blood clearance (ABC) phenomenon. Complement activation is well known to play an important role in the clearance of PEGylated and non-PEGylated nanomedicines following intravenous injection. This complement system is also thought to be responsible for the ABC phenomenon wherein repeated injections of PEGylated products are bound by anti-PEG antibodies. This study used three different sources of anti-PEG antibodies: HIK-M09 monoclonal antibodies (mAbs); HIK-M11 mAbs; and antiserum containing polyclonal anti-PEG IgMs. 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-n-[methoxy (polyethylene glycol)-2000] (mPEG2000-DSPE) was immobilized as an antigen on aminopropyl silane biosensor chips of BLI. All anti-PEG IgMs in the sources increased the signals (thickness of the layer around the sensor tip) regarding binding of anti-PEG antibodies to PEG on the chips. In all anti-PEG IgM sources, further increases in the signals were observed when incubated in naïve mouse serum, which is a complement source, but not in heat inactivated (56 °C, 30 min) mouse serum, which abolishes complement activity. These findings show that the complement activation mediated via anti-PEG IgMs, which occurred on the sensor chips, was detected via BLI analysis. The complement activation induced by all anti-PEG IgM sources was confirmed via conventional enzyme-linked immunosorbent assay (ELISA), which is the conventional mode for detection of complement activation. Our study results show that BLI is a simple alternative method for the detection of complement activation.

Anti-PEG IgM production and accelerated blood clearance phenomenon after the administration of PEGylated exosomes in mice.

Recently, there is an increasing interest in exosomes or extracellular vesicles as potential candidates for delivering RNAs, proteins, genes, and anticancer agents. Engineering of exosome properties is rapidly evolving as a means of expanding exosome applications. PEGylation of exosomes is a technique used to improve their in vivo stability, circulation half-lives, and sometimes to allow the binding targeting ligands to the exosome exterior. According to FDA guidelines for the development of PEGylated proteins, immunological responses to PEGylated molecules and particles should be examined. In this study, we prepared PEGylated exosomes and investigated the production of anti-PEG IgM antibodies after single i.v. injections in mice. In addition, we monitored blood concentrations and tumor accumulation of a second dose of PEGylated exosomes administered after the initial dose. Single injections of PEGylated exosomes in mice induced anti-PEG IgM production in a T cell-dependent manner. The anti-PEG IgM production decreased when the injection dose of PEGylated exosomes was further increased. Anti-PEG IgM induced by injection of PEGylated exosomes decreased blood concentrations of a second dose of PEGylated exosomes and suppressed their tumor accumulation in a C26 murine colorectal cancer model. Initial injection doses of either PEGylated liposomes or PEGylated ovalbumin (PEG-OVA), both of them induced anti-PEG IgM production, also decreased the blood concentration of PEGylated exosomes. Interestingly, anti-PEG IgM induced by injection of PEGylated exosomes did not affect the blood concentration of PEG-OVA. These results imply the importance of monitoring anti-PEG IgM when repeat PEGylated exosome doses are required and/or when PEGylated exosomes are used together with other PEGylated therapeutics.

Incorporating Gangliosides into PEGylated Cationic Liposomes that Complexed DNA Attenuates Anti-PEG Antibody Production but Not Anti-DNA Antibody Production in Mice.

Gangliosides (glycosphingolipids) reduce antibody production by inhibiting B-cell receptor (BCR) signaling. We have shown that a copresentation of gangliosides and polyethylene glycol (PEG) on the same liposomes suppresses anti-PEG IgM production in mice. In addition, we recently observed that pDNA incorporated in PEGylated cationic liposomes (PCLs) induces anti-DNA IgM, which could be a hurdle to the development of efficient gene delivery systems. Therefore, the focus of this study was to determine if the copresentation of gangliosides and DNA on the same PCL would suppress antibody production against DNA. PCLs including DNA induced both anti-PEG IgM production and anti-DNA IgM production. The extent of anti-PEG and anti-DNA IgM production was likely dependent on the immunogenicity of the complexed DNA. Treatment of clodronate-containing liposomes, which causes a depletion of phagocytic cells, suppressed anti-PEG IgM production from PCLs that did not include DNA but failed to suppress anti-PEG IgM production from PCLs that complexed DNA (PCLD). Both anti-PEG IgM and anti-DNA IgM was induced in T-cell-deficient nude mice as well as in normal mice following treatment with PCLs and PCLD, respectively. These results indicate that phagocytic cells contribute to anti-PEG IgM production but not to anti-DNA IgM production, while T-cells do not contribute to any form of antibody production. The copresentation of gangliosides and DNA significantly reduced anti-PEG IgM production but unfortunately did not reduce anti-DNA IgM production. It appears that the immunosuppressive effect of gangliosides, presumably via the CD22 signaling pathway, is limited only to anti-PEG immunity.

Nucleic acids delivered by PEGylated cationic liposomes in systemic lupus erythematosus-prone mice: A possible exacerbation of lupus nephritis in the presence of pre-existing anti-nucleic acid antibodies.

Nucleic acid-based therapy with plasmid DNA (pDNA) and small interfering RNA (siRNA) have received recent attention for their ability to modulate the cellular expression of genes and proteins. Polyethylene glycol-modified (PEGylated) cationic nanoparticles have been used as non-viral vectors for the in vivo delivery of these nucleic acids. We have reported that PEGylated cationic liposomes (PCL) including pDNA or siRNA induce anti-PEG antibodies upon repeated intravenous injection, leading to the formation of immune complexes and enhanced clearance from the blood of subsequent doses. However, the issue surrounding the association of nucleic acids with PCL whether induces anti-nucleic acid antibodies has not been studied. Systemic lupus erythematosus (SLE) is a chronic autoimmune disease with the character of end-organ damage and the presence of anti-nuclear antibodies. We used a healthy mouse and an SLE mouse model to test the hypothesis that nucleic acids associated with PCL induce anti-nuclear antibodies and then induce SLE and exacerbate SLE symptoms. We report here that pDNA or siRNA associated with PCL (pDNA/PCL or siRNA/PCL) induced anti-DNA or RNA antibodies, respectively, in healthy mice. Repeated injections did not, however, cause SLE-like symptoms in the healthy mice. In addition, in SLE-prone mice with pre-existing anti-nuclear antibodies, pDNA/PCL were deposited on the kidneys and exacerbated lupus nephritis subsequent to the formation of immune complexes. These results may imply that nucleic acids associated with PCL do not contribute to the onset of SLE in healthy individuals who lack anti-nuclear antibodies, but nucleic acids may exacerbate the symptoms in SLE patients who have pre-existing anti-nuclear antibodies.

Pegfilgrastim (PEG-G-CSF) Induces Anti-polyethylene Glycol (PEG) IgM via a T Cell-Dependent Mechanism.

Protein-based therapeutics are beginning to be widely used in various clinical settings. Conjugation of polyethylene glycol (PEGylation) to protein therapeutics improves their circulation half-lives in the body. However, we and other groups observed that the initial dose of some PEGylated protein-based therapeutics may induce anti-PEG antibodies (primarily immunoglobulin M (IgM)), resulting in the accelerated clearance of a second dose. The mechanism behind the induction of anti-PEG IgM by PEGylated protein-based therapeutics is still unclear. In this study, we found that Pegfilgrastim (PEG-G-CSF, the PEGylated form of the recombinant human granulocyte colony-stimulating factor) induced anti-PEG IgM in mice when administered via either intravenous or subcutaneous administration. However, the anti-PEG IgM induction is diminished both in athymic nude mice lacking T cells and in splenectomized mice. In addition, anti-PEG IgM production was significantly diminished in the cyclophosphamide-treated mice depleted of B-cells. These results indicate that anti-PEG IgM production by Pegfilgrastim occurs in spleen in a T cell-dependent manner, which differs from anti-PEG IgM induced by PEGylated liposomes. However, B cells, both marginal zone and follicular, are essential for anti-PEG IgM production in both PEGylated preparations.

Impact of Pre-Existing or Induced Anti-PEG IgM on the Pharmacokinetics of Peginterferon Alfa-2a (Pegasys) in Mice.

PEGylation had been used successfully to improve the circulation half-lives and some physicochemical properties of protein therapeutics. However, anti-polyethylene glycol (anti-PEG) antibodies, either pre-existing or treatment-induced, can negatively affect the pharmacokinetics and pharmacological efficacy of PEGylated proteins. We have examined anti-PEG immune responses in mice for peginterferon alfa-2a (Pegasys), a clinically approved PEGylated protein therapeutic, at both the recommended dose (equivalent to 3 µg/kg in mice) and at higher doses (150 µg/kg) for single or repeated subcutaneous (s.c.) administrations. The effect of treatment-induced anti-PEG IgM on serum concentrations of Pegasys, following repeated administrations, was evaluated. In addition, the effect of pre-existing anti-PEG IgM elicited by a different PEGylated protein, PEG-OVA, on the systemic clearance of Pegasys, was investigated. At a s.c. dose of 3 µg/kg, single injections of Pegasys barely elicited anti-PEG immune responses. Four repeated doses of 150 µg/kg Pegasys elicited anti-PEG IgM production, depending on dose frequency, and triggered the rapid clearance of subsequent doses. In addition, anti-PEG-IgM produced in response to prior administration of PEG-OVA caused a rapid blood clearance of Pegasys. Our results, therefore, underscore the importance of screening for both pre-existing and treatment-induced anti-PEG antibodies in patients prior to and during treatment with PEGylated protein drugs.

Pegfilgrastim (PEG-G-CSF) induces anti-PEG IgM in a dose dependent manner and causes the accelerated blood clearance (ABC) phenomenon upon repeated administration in mice.

Pegfilgrastimis a recombinant PEGylated human granulocyte colony-stimulating factor (G-CSF) analog filgrastim (trade names Neulasta® or G-Lasta®) that stimulates the production of white blood cells (neutrophils). It is employed as an alternative to filgrastim (G-CSF) for chemotherapy-induced neutropenia in patients due to its longer half-life. In clinical settings, PEG-G-CSF is administered to cancer patients via both the s.c. and i.v. routes. In a murine study, we showed that, regardless of administration route, initial doses of PEG-G-CSF above 0.06 mg/kg elicited anti-PEG immune response in a dose-dependent manner. I.v. administration elicited higher levels of anti-PEG IgM than the s.c. route. Initial doses of PEG-G-CSF (6 mg/kg) that were high enough to trigger production of anti-PEG IgM, did not trigger the accelerated clearance of a lower subsequent dose (0.06 mg/kg) that was similar to i.v. clinical doses of PEG-G-CSF, but when the subsequent dose of PEG-G-CSF was raised to (6 mg/kg), the initial dose triggered the accelerated clearance of the second dose via an anti-PEG IgM-mediated complement activation. Similar observations were noted when an increased PEG-OVA dose was given as the second dose, indicating that pre-existing and/or treatment-induced anti-PEG antibodies might compromise the therapeutic activity and/or reduce tolerance of other PEGylated formulations. To the best of our knowledge, this is the first report to suggest the induction of the ABC phenomenon upon repeated injections of pegfilgrastim. In the clinic, cancer patients, receiving multiple cycles of chemotherapy, receive multiple cycles of pegfilgrastim to avoid infections and substantial morbidity. The ABC phenomenon to pegfilgrastim appears to be the cause of loss of clinical benefit of sequential treatments with pegfilgrastim in patients.

Hepatosplenic phagocytic cells indirectly contribute to anti-PEG IgM production in the accelerated blood clearance (ABC) phenomenon against PEGylated liposomes: Appearance of an unexplained mechanism in the ABC phenomenon.

The accelerated blood clearance (ABC) phenomenon, caused in large degree via in vivo anti-PEG IgM production, is one of obstacles for development of PEGylated liposome and protein formulations, due to decreased efficiency and/or side effects such as anaphylaxis upon repeat administrations. We have shown in murine ABC models that splenectomy suppressed the level of anti-PEG IgM production induced by PEGylated liposomes, indicating that murine splenic B cells play an important role in its production. However, splenectomy did not completely inhibit production of anti-PEG IgM, suggesting that other cells may contribute to its production in the ABC phenomenon. In this study, we examined the contribution of hepatosplenic phagocytic cells to anti-PEG IgM production and clearance of PEGylated liposomes during the ABC phenomenon. Depletion of hepatosplenic phagocytic cells by pretreatment of mice with clodronate-containing non-PEGylated liposomes suppressed anti-PEG IgM production to a considerable degree, without a change in the number of splenic B cells, and attenuated the enhanced clearance of second dose of PEGylated liposomes. These results suggest that hepatosplenic phagocytic cells, in addition to splenic B cells, contribute to the production of anti-PEG IgM and the ABC phenomenon against PEGylated liposomes. The mechanism whereby splenic B cells interact with hepatosplenic phagocytic cells to produce anti-PEG IgM, upon administration of an initial dose of PEGylated liposomes remains to be elucidated.

Cancer cell-type tropism is one of crucial determinants for the efficient systemic delivery of cancer cell-derived exosomes to tumor tissues.

Exosomes are gaining increasing attention as drug delivery vehicles due to their low toxicity and ability to functionally transfer biological cargos between cells. However, the therapeutic applicability of exosomes is partially hampered by a lack of cell-type specificity. In this study, therefore, we investigated the impact of cell-type tropism on the in vivo systemic delivery of exosomes to tumor tissues. Exosomes derived from murine colorectal cancer cells (C26) (C26-Exos) and murine melanoma cells (B16BL6) (B16BL6-Exos) were collected. In vitro cellular uptake of either autologous (C26) or allogeneic (B16BL6) exosomes by C26 tumor cells was determined. In vivo tumor accumulation of each type of exosomes in mice bearing C26 tumors was monitored with an in vivo imaging system (IVIS). In in vitro studies, autologous C26-Exos were more efficiently taken up by C26 cancer cells, compared to allogeneic B16BL6-Exos. For in vivo studies, exosomes were modified with surface polyethylene glycol (PEG) to improve their circulation lifetimes. Although both types of PEGylated exosomes accumulated in C26-tumor tissue, autologous exosomes were preferentially accumulated within C26-tumor tissue compared to allogeneic exosomes. The increased tumor accumulation of autologous PEGylated exosomes was accompanied by the preferential uptake of exosomes by not only C26-tumor cells but also tumor-associated immune cells. This study implies that cancer cell-type tropism is an important factor in the achievement of tumor cell targeting with cancer cell-derived exosomes.