Bioactive telodendrimer nanodrug for immune modulation and drug delivery

Abstract

Disclosed herein are novel well-defined telodendrimer nanodrugs featured with multiple negative charges and hydrophobic groups to mimic endotoxin for toll-like receptor binding, thus to modulate immune response. The next generation TD nanodrugs can actively fine-tuned inflammation control against a broad spectrum of immune stimuli via pleiotropic mechanisms; and some TD nanodrugs can stimulate immune response by activating TLR for potential cancer immunotherapy; as well as TD nanodrugs that can form nanocarrier for the delivery of vital antibiotics, anticancer drugs or other therapeutics for disease treatments with simultaneous immune modulation effects.

Description

BIOACTIVE TELODENDRIMER NANODRUG FOR IMMUNE MODULATION AND DRUG DELIVERYCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to US Provisional Application No. 63 / 471 ,646, filed on June 7, 2023, the entire disclosures of which are incorporated herein by reference as though set forth in full.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under grant number GM 130941 awarded by the National Institute of Health. The government has certain rights in the invention.BACKGROUND

[0001] Sepsis is the body’s dysregulated immune response to systemic infections caused by pathogens such as bacteria, viruses, or fungi. This is a lifethreatening health emergency that attacks about 1.7 million adults in the US and causes about 350,000 deaths annually. In sepsis, our innate immune cells can recognize pathogen-associated molecular patterns (PAMPs) and damage- associated molecular patterns (DAMPs) as nonself-danger signals through pattern recognition receptors (PRRs) signals. When inflammatory responses are not resolved, a cytokine storm occurs which is a combination of an excessive pro- inflammatory response and an inadequate anti-inflammatory response, leading to the disruption of the immune system's homeostasis. This can cause hemodynamic instability, tissue damage, disseminated intravascular coagulation (DIC), organ failure, and ultimately death. PAMPs are exclusive to host cells and include endotoxin from Gram-negative (GN) bacteria, lipoteichoic acid (LTA) from Grampositive (GP) bacteria, bacterial or viral nucleic acids (DNA or RNA), and more. DAMPs are released during tissue injury or cell death and include histones, high- mobility group box 1 (HMGB1) protein, and more. Disease progression is driven by simultaneous derangements of multiple pathways. Despite advanced care along with nearly 23.7 billion dollars in hospital expenses per year, one in every three people who dies in a hospital had sepsis during hospitalization (a recent CDC report).

[0002] Gram-negative (GN) bacterial systemic infection is responsible for more than 60 percent of cases of severe sepsis. During GN bacteremia, free Lipopolysaccharide (LPS) spread in blood circulation binds to Toll-like receptors (e.g. TLR-4) on immune cell surfaces (e.g., macrophages, dendritic cells) with the help of LPS-binding-protein (LBP) and MD-2 co-receptor. Upon ligand recognition, TLR4 will undergo dimerization and endocytosis followed by downstream signaling via either MyD88-dependent or TRIF-dependent pathways. MyD88-dependent pathway activates phosphorylation and nuclear translocation of nuclear factor-KB (NF-KB) and AP-1 , initiating the transcription of pro-inflammatory cytokine genes (e.g., TNF-a, IL- 1 , and IL-6). This will trigger inflammatory responses against the pathogens to control the infection and maintain the normal physiological condition. However, the onset of sepsis is linked to an overactive immune response to external stimuli, so it is essential to resolve this reaction for successful treatment. Current sepsis therapies include broad-spectrum antibiotics along with fluid resuscitation and vasopressors to control early infections and support organ perfusion. Although these antibiotics can kill the bacteria they are unable to neutralize the released endotoxin which activates secondary immune responses. On top of that, infections caused by multi-drug resistant (MDR) GN bacteria make it more difficult to treat.

[0003] Peptide antibiotic Polymyxin B is a polycationic antimicrobial and is one of the most potent LPS binders. PMB can bind to microbial cell envelopes through its strong positive charge, and rupture the cell membrane causing leakage of the cell contents. In addition, the high affinity of PMB to LPS can neutralize released endotoxin which subsequently inhibits systemic inflammation and controls organ dysfunction. Such dual capacity of PMB makes it a highly efficient arsenal against MDR GN pathogens. Unfortunately, its application as first-line therapy during the early stage of sepsis treatment is limited due to the negative side effects such as nephrotoxicity, neuro-, and hepatotoxicity. Indeed, poor stability of PMB in the bloodstream increases the high dose requirement which is associated with their dose-limiting cytotoxicity. Again, the high binding affinity of PMB to megalin (found on renal proximal tubular cells) increases their kidney accumulation during systemic administration which exhibits renal toxicity. Although several attempts have been taken to find a less-toxic derivative of PMB, there remains an absence of appropriate therapies. Thus, there remains a need for improved systemic delivery of antibiotics,as well as new drug therapies that can inhibit immune cell inflammation stimulated by different immunogenic molecules.SUMMARY OF THE INVENTION

[0004] Embodiments disclosed herein are directed to immune modulating nanodrugs having the formula: P-B(X)-(R®m-L-Rn)x, wherein P is a hydrophilic oligomer or polymer; B(X) is a branching unit; R® is at least one negatively charged moiety and all R® are the same; L is an optional linker group or a bond; R is at least one hydrophobic moiety; m is integer from 1-16; n is integer from 1-16; and x is integer from 1-8.

[0005] According to embodiments disclosed herein, P is a polyethylene glycol (PEG) polymer having an average molecular weight from 44 Da to 40 kDa, or a dendritic zwitterionic polymer, or a dendritic zwitterionic glycerol phosphorylcholine polymer. According to embodiments, B(X) branching unit is at least one amino acid having more than one amine group, and can be an amino acid selected from lysine, ornithine, 2,3-bisaminopropanoic acid, 3-(Bis(3-aminopropyl)amino)propanoic acid, 4,5-bisaminopentanoic acid, 2,4-bisaminobutanoic acid, 3,4-bisaminobutanoic acid, and 3,5-Bis(aminomethyl)-benzoic acid). According to embodiments, R® is a negatively charged acid, and can be one of the following acids: carboxylic acid, succinic acid, itaconic acid (ITA), squaric acid, oxalic acid, sulfuric acid, or phosphoryl acid (PA). According to embodiments, L can be polyethylene glycol moiety, an amino acid, a polyamino acid, a polyserine moiety, an enzyme cleavable peptide moiety, a disulfide bond moiety and an acid labile moiety, a polyglycine moiety, a poly(serine-glycine) moiety, an aliphatic amino acid moiety, a 6-amino hexanoic acid moiety, a 5-amino pentanoic acid moiety, a 4-amino butanoic acid moiety, a beta-alanine moiety, or a combination of the aforementioned.

[0006] According to embodiments, R is a saturated fatty acid or an unsaturated fatty acid, or one of the following: ethyl (C2) , propyl (C3), butyl (C4), pentyl (C5), hexyl (C6), heptyl (C7) t2-propypentanoyl (PPA), 4-phenylbutyl (PBA), nonanoyl (C9), heptadecanoyl (C17), lauroyl (C12), myristoyl (C14), palmitoyl (C16), oleoyl (C18), cholesteryl (CHO), vitamin E succinyl (VE), riboflavin glutaryl (Rf), cholic acetyl (CA), or fluvastatin (Flu).

[0007] According to embodiments, the immune modulating nanodrugs disclosed herein provide immune modulating activity by altering immune cell response in infection and inflammation. According to embodiments disclosed herein, are methods of modulating immune responses in a mammal by that inhibit the activation of a Toll-like receptors (TLRs) and the method comprising administering a therapeutically effective amount of the nanodrugs disclosed herein. According to an embodiment, the nanodrug inhibits Toll-like receptor signaling stimulated by damage associated molecular patterns (DAMPs) and pathogen associated molecular patterns (PAMPs).

[0008] According to embodiments disclosed herein, some of the immune modulating nanodrug have the following formula: PEG5kSUA8C174, PEG5kSA8C174, PEG5kOA8C174, PEG5kPA8C174, PEG5kSUA8C124, PEG5kSUA8C144, PEG5kSUA8C164, PEG5kSUA8C184, PEG5kSUA8CHO4, PEG5kSUA8VE4, PEG5kSUA8Rf4, PEG5kSUA8FIU4, PEG20kSUA8C174, PEG2kSUA8C174, GPC8SUA8C174, PEG5kSUA8-L-C174, PEG5kSUA4C174, PEG5kSA4C174, PEG5kOA4C174, PEG5kPA4C174, PEG5kITA4, PEG5kITA8, PEG5kITA8C174, or PEG5kITA8-L-C174.

[0009] According to embodiments disclosed herein, are methods of modulating an immune response in a mammal by binding and activating a Toll-like receptor. The method comprises the step of administering a therapeutically effective amount of ta nanodrug disclosed herein. According to embodiments this method utilizes one or a combination of the following nanodrugs: PEG5kSUA8C44, PEG5kSUA8PPA4, PEG5kSUA8PBA4, PEG5kSUA6C176, or PEG2kSUA6C176.

[0010] According to embodiments, are methods of modulating an immune response in a mammal by inhibiting the activity of cytokine for cytokine receptor activation by administering a therapeutically effective amount of a nanodrug disclosed herein. Also disclosed are embodiments directed to methods of modulating an immune response in a mammal by inhibiting cytokine activity via preventing the oligomerization of cytokine receptors by interrupting the fluidity of the receptor-residing lipid raft by administering a therapeutically effective amount of a nanodrug disclosed herein.

[0011] According to embodiments disclosed herein are methods of modulating an immune response in a mammal by administering a therapeutically effective amount of a nanodrug disclosed herein, which stimulate immune cells that inhibit tumor necrosis factor-alpha (TNF-a) and lnterleukin-1 (I L-1 b) activity in a mammal in need thereof. Some of the disclosed nanodrugs for use in this method are as follows: PEG5kSUA8C174, PEG5kSA8C174, PEG5kOA8C174, PEG5kPA8C174, PEG5kSUA8C184, PEG5kSUA8CHO4, PEG5kSUA8VE4, PEG20kSUA8C174, PEG2kSUA8C174, GPCSSUA8C174, PEG5kSUA8-L-C174, PEG5kSUA4C174, PEG5kSA4C174, PEG5kOA4C174, and PEG5kPA4C174.

[0012] Further embodiments disclosed herein include a nanocarrier composition comprising multiple self-assembled units with the formula: P-B(X)-(R®m- -L-Rn)x, wherein P is a hydrophilic oligomer or polymer; B(X) is a branching unit; R® is at least one negatively charged moiety and all R® are the same; L is an optional linker group or a bond; R is at least one hydrophobic moiety; m is integer from 1-16; n is integer from 1-16; and x is integer from 1 -8.

[0013] According to embodiments, the disclosed nanocarrier compositions comprises at least one therapeutic compound. According to embodiments the nanocarrier compositions encapsulate at least one positively charged therapeutic compound selected from the group consisting of: an antibiotic peptide, a lysozyme, a cytocrhom c, a growth factor, a fibroblast growth factor (FGF), vascular endothelium growth factor (VEGF), an interlukin-4 (IL-4), an interlukin-10 (IL-10), a interferon-r (INF-r), a tumor growth factor- (TGF- ), a chemokine, or an interlukin-8 (IL-8). According to other embodiments the nanocarrier compositions encapsulate at least one therapeutic compound from the group: polymyxin B, polymyxin A, aminoglycosides, gentamycin, Daptomycin, or doxorubicine, In further embodiments the nanocarrier compositions encapsulates therapeutic hydrophobic compounds, such as anticancer drug, an antibiotic, an anti-inflammatory drug, paclitaxel, docetaxel, curcumin, imipenem, doxycycline, ceftaroline, methyl itaconate (ITA), or erythomycin. In still other embodiments, the nanocarrier compositions chelate platinum drug compounds, such as, cisplatin, carboplatin, or oxaliplatin.

[0014] Further embodiments include methods of modulating an immune response for the treatment of bacterial infection, inflammation, sepsis and cancer, ina mammal in need thereof by administering a therapeutically effective amount of the disclosed nanocarrier compositions.

[0015] The embodiments disclosed herein demonstrate the in vitro safety and efficiency of the Nano-PMB for direct killing of GN bacteria and inactivating broadspectrum septic molecules (e.g., PAMPs and DAMPs) in both mice and human cell lines. The nanoformulations are evaluated for in vivo toxicity and subjected to the CLP model (Cecum Ligation and Puncture (CLP)-lnduced Sepsis Model) in mice, which involves polymicrobial infection and thus represents a clinically relevant septic state. This novel Nano-PMB is promising for clinical translation in the treatment of GN sepsis by effective control of both infection and hyperinflammation.BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1.1 presents structures of telodendrimers with anti-inflammatory and inflammation induction properties, including PEGnXAnRn, PEGnXAn-L-Rn, PEGnXAn. XA can be Succinic acid (SUA), Squaric acid (SA), Oxalic acid (OA), Phosphoryl acid (PA), Itaconic acid (ITA). R group can be Acetyl (OAc), butyryl (C4), 2-Propypentanoyl (PPA), 4-Phenylbutyl (PBA), Nonanoyl (C9), Heptadecanoyl (C17), Lauroyl (C12), Myristoyl (C14), Palmitoyl (C16), Oleoyl (C18), Cholesteryl (OHO), Vitamin E succinyl (VE), Riboflavin glutaryl (Rf), Cholic acetyl (CA), Fluvastatin (Flu). L can be PEG470-linker, 6-aminohexanoic acid, and 3,4- Diaminobutanoic acid.

[0017] Figure 1 .2 presents the synthesis route of PEGnR’sR4. R’ can be Succinic acid (SUA), Squaric acid (SA), Oxalic acid (OA), Phosphoryl acid (PA), Itaconic acid (ITA). R group can be Acetyl (OAc), butyryl (C4), 2-Propypentanoyl (PPA), 4- Phenylbutyl (PBA), Nonanoyl (C9), Heptadecanoyl (C17), Lauroyl (C12), Myristoyl (C14), Palmitoyl (C16), Oleoyl (C18), Cholesteryl (CHO), Vitamin E succinyl (VE), Riboflavin glutaryl (Rf), Cholic acetyl (CA), Fluvastatin (Flu).

[0018] Figure 1 .3 presents the synthesis route of PEGnR’4R4. R’ can be Succinic acid (SUA), Squaric acid (SA), Oxalic acid (OA), Phosphoryl acid (PA), Itaconic acid (ITA). R group can be Acetyl (OAc), butyryl (C4), 2-Propypentanoyl (PPA), 4- Phenylbutyl (PBA), Nonanoyl (C9), Heptadecanoyl (C17), Lauroyl (C12), Myristoyl (C14), Palmitoyl (C16), Oleoyl (C18), Cholesteryl (CHO), Vitamin E succinyl (VE), Riboflavin glutaryl (Rf), Cholic acetyl (CA), Fluvastatin (Flu).

[0019] Figure 1.4 presents the synthesis route of PEGnR’s-L-R4. R’ can be Succinic acid (SUA), Squaric acid (SA), Oxalic acid (OA), Phosphoryl acid (PA), Itaconic acid (ITA). R group can be Acetyl (OAc), butyryl (C4), 2-Propypentanoyl (PPA), 4-Phenylbutyl (PBA), Nonanoyl (09), Heptadecanoyl (C17), Lauroyl (C12), Myristoyl (C14), Palmitoyl (C16), Oleoyl (C18), Cholesteryl (CHO), Vitamin E succinyl (VE), Riboflavin glutaryl (Rf), Cholic acetyl (CA), Fluvastatin (Flu). L can be PEG470- linker, 6-aminohexanoic acid, and 3,4-Diaminobutanoic acid.

[0020] Figure 1 .5 presents the synthesis route of R’sR4. R’ can be Succinic acid (SUA), Squaric acid (SA), Oxalic acid (OA), Phosphoryl acid (PA), Itaconic acid (ITA). R group can be Acetyl (OAc), butyryl (C4), 2-Propypentanoyl (PPA), 4-Phenylbutyl (PBA), Nonanoyl (C9), Heptadecanoyl (C17), Lauroyl (C12), Myristoyl (C14), Palmitoyl (C16), Oleoyl (C18), Cholesteryl (CHO), Vitamin E succinyl (VE), Riboflavin glutaryl (Rf), Cholic acetyl (CA), Fluvastatin (Flu).

[0021] Figure 1 .6 presents the synthesis route of PEGnR’eR6. R’ can be Succinic acid (SUA), Squaric acid (SA), Oxalic acid (OA), Phosphoryl acid (PA), Itaconic acid (ITA). R group can be Acetyl (OAc), butyryl (C4), 2-Propypentanoyl (PPA), 4- Phenylbutyl (PBA), Nonanoyl (C9), Heptadecanoyl (C17), Lauroyl (C12), Myristoyl (C14), Palmitoyl (C16), Oleoyl (C18), Cholesteryl (CHO), Vitamin E succinyl (VE), Riboflavin glutaryl (Rf), Cholic acetyl (CA), Fluvastatin (Flu).

[0022] Figure 1.6A presents the synthesis route of PEGnR’2Re. R’ can be Succinic acid (SUA), Squaric acid (SA), Oxalic acid (OA), Phosphoryl acid (PA), Itaconic acid (ITA). R group can be Acetyl (OAc), butyryl (C4), 2-Propypentanoyl (PPA), 4-Phenylbutyl (PBA), Nonanoyl (C9), Heptadecanoyl (C17), Lauroyl (C12), Myristoyl (C14), Palmitoyl (C16), Oleoyl (C18), Cholesteryl (CHO), Vitamin E succinyl (VE), Riboflavin glutaryl (Rf), Cholic acetyl (CA), Fluvastatin (Flu).

[0023] Figure 1 .7 presents the synthesis route of PEGnR’2Re. R’ can be Succinic acid (SUA), Squaric acid (SA), Oxalic acid (OA), Phosphoryl acid (PA), Itaconic acid (ITA). R group can be Acetyl (OAc), butyryl (C4), 2-Propypentanoyl (PPA), 4- Phenylbutyl (PBA), Nonanoyl (C9), Heptadecanoyl (C17), Lauroyl (C12), Myristoyl (C14), Palmitoyl (C16), Oleoyl (C18), Cholesteryl (CHO), Vitamin E succinyl (VE), Riboflavin glutaryl (Rf), Cholic acetyl (CA), Fluvastatin (Flu).

[0024] Figure 1.8 presents the synthesis route of PEGnR’4. R’ can be Succinic acid (SUA), Squaric acid (SA), Oxalic acid (OA), Phosphoryl acid (PA), Itaconic acid (ITA).

[0025] Figure 1.9 presents the synthesis route of PEGnR’s. R’ can be Succinic acid (SUA), Squaric acid (SA), Oxalic acid (OA), Phosphoryl acid (PA), Itaconic acid (ITA).

[0026] Figure 1.10 presents the chemical structures of TD and oligomer examples, include anti-inflammatory TDs: Figure 1.10A presents PEG5kSUA8OAc4, PEG5kSUA8C44, PEG5kSUA8PPA4, PEG5kSUA8PBA4, PEG5kSUA8C94, PEG5kSUA8C174, PEG5kSA8C174, PEG5kOA8C174, PEG5kPA8C174, PEG5kSUA8C124, PEG5kSUA8C144, PEG5kSUA8C164, PEG5kSUA8C184, PEG5kSUA8CHO4, PEG5kSUA8VE4, PEG5kSUA8Rf4, PEG5kSUA8CA4, PEG5kSUA8Flu4, PEG20kSUA8C174, PEG2kSUA8C174, GPCSSUA8C174, PEG5kSUA8-L-C174, PEG5kSUA4C174, PEG5kSA4C174, PEG5kOA4C174, PEG5kPA4C174, PEG5kITA4, PEG5kITA8, PEG5kITA8C174, PEG5kITA8-L-C174; anti-inflammatory oligomers; Figure 1.10B presents SUA8OAc4, SUA8C44, SUA8PPA4, SUA8PBA4, SUA8C94, SUA8C174; and proinflam matory TDs: and Figure 1.10C presents PEG2kSUA8C176, PEG5kSUA6C17e, PEG5kPA2C96, PEG5kPA2C126and

[0027] Figure 1.11 presents cell viability of HEK 293 cell incubated with TD examples at 1000, 500, 100, 50 pg / mL for 72 h. TDs include PEG5kSUA8OAc4, PEG5kSUA8C44, PEG5kSUA8PPA4, PEG5kSUA8PBA4, PEG5kSUA8C94, PEG5kSUA8C174, PEG5kSA8C174, PEG5kOA8C174, PEG5kPA8C174, PEG5kSUA8C124, PEG5kSUA8C144, PEG5kSUA8C164, PEG5kSUA8C184, PEG5kSUA8CHO4, PEG5kSUA8VE4, PEG5kSUA8Rf4, PEG5kSUA8CA4, PEG5kSUA8Flu4, PEG20kSUA8C174, PEG2kSUA8C174, GPC8SUA8C174, PEG5kSUA8-L-C174, PEG5kSUA4C174, PEG5kSA4C174, PEG5kOA4C174, PEG5kPA4C174, PEG5kITA4, PEG5kITAs, PEG5kITA8C174, PEG5kITA8-L-C174, PEG2kSUA6C176, PEG5kSUA6C176,PEG5kPA2C96, PEG5kPA2C126.

[0028] Figure 1.12 presents cell viability of THP-1 cell incubated with TD examples at 1000, 500, 100, 50 pg / mL for 72 h. TDs include PEG5kSUA8OAc4, PEG5kSUA8C44, PEG5kSUA8PPA4, PEG5kSUA8PBA4, PEG5kSUA8C94, PEG5kSUA8C174, PEG5kSA8C174, PEG5kOA8C174, PEG5kPA8C174, PEG5kSUA8C124, PEG5kSUA8C144, PEG5kSUA8C164, PEG5kSUA8C184, PEG5kSUA8CHO4, PEG5kSUA8VE4, PEG5kSUA8Rf4, PEG5kSUA8CA4, PEG5kSUA8Flu4, PEG20kSUA8C174, PEG2kSUA8C174, GPCSSUA8C174, PEG5kSUA8-L-C174, PEG5kSUA4C174, PEG5kSA4C174, PEG5kOA4C174, PEG5kPA4C174, PEG5kITA4, PEG5kITA8,PEG5kITA8C174, PEG5kITA8-L-C174, PEG2kSUA6C176, PEG5kSUA6C178, PEG5kPA2C96, PEG5kPA2C126.

[0029] Figure 1 .12A presents cell viability of RAW 264.7 cell incubated with TD examples at 1000, 500, 100, 50 pg / mL for 72 h. TDs include PEG5kSUA8OAc4, PEG5kSUA8C44, PEG5kSUA8PPA4, PEG5kSUA8PBA4, PEG5kSUA8C94, PEG5kSUA8C174, PEG5kSA8C174, PEG5kOA8C174, PEG5kPA8C174, PEG5kSUA8C124, PEG5kSUA8C144, PEG5kSUA8C164, PEG5kSUAsC184, PEG5kSUA8CHO4, PEG5kSUA8VE4, PEG5kSUA8Rf4, PEG5kSUA8CA4, PEG5kSUA8Flu4, PEG20kSUA8C174, PEG2kSUA8C174, GPC8SUA8C174, PEG5kSUA8-L-C174, PEG5kSUA4C174, PEG5kSA4C174, PEG5kOA4C174, PEG5kPA4C174, PEG5kITA4, PEG5kITA8, PEG5kITA8C174, PEG5kITA8-L-C174, PEG2kSUA6C176, PEG5kSUA6C176,PEG5kPA2C9e, PEG5kPA2C12e. Figure 1.12B presents cell viability of RAW 264.7 cell incubated with TD examples at 1000, 500, 100, 50 pg / mL for 24 h. TDs include PEG5kSUAsC174, PEG5kSA4C174, PEG5kOA4C174, PEG5kPA4C174, PEG5kSUA8CHO4, PEG5kOA4C174, PEG5kITA8-L-C174.

[0030] Figure 1.13 presents a heatmap of IC50 (pM) of TDs for 50 ng / mL LPS induced TNF-a production in RAW 264.7 and THP-1 cells, 100 ng / mL LPS activated HEK-TLR-4 and THP-1 Blue cells, and 1 pg / mL LTA activated HEK-TLR-2 cells.

[0031] Figure 1.14 presents proinflammatory cytokines TNF-a and IL-6 production in sepsis patient induced by 10 ng / mL LPS for 2 h. (A) TNF-a and IL-6 production in sepsis patient induced by 10 ng / mL LPS incubated with PEG5kSUA8C174, PEG5kSUA8CHO4, and PEG5kPA8C174at 25, 50 and 100 pg / mL for 2 h. (B) TNF-a and IL-6 production in sepsis patient induced by 10 ng / mL LPS incubated with free ITA and ITA-PEG5kSUA8C174at concentration of 50 pg / mL ITA and 500 pg / mL PEG5kSUA8C174for 2 h.

[0032] Figure 1.15 Phagocytosis of E.Coli and Staph, aureus in RAW 264.7 cells. Phagocytosis of E.Coli (Figure 1 .15A) and Staph. Aureus (Figure 1.15B) of RAW 264.7 cells incubated together with different concentration of PEG5kSUA8C174for 8 h. Phagocytosis of Staph. Aureus (Figure 1.15C) and E.Coli (Figure 1.15D) of LPS activated RAW 264.7 cells incubated together with 100 pg / mL PEG5kSUA8C174, 10 pg / mL free gentamicin (GM), and 10:100 pg / mL GM- PEG5kSUA8C174for overnight pretreatment.

[0033] Figure 1.16 Cellular uptake of PEG5kSUA8C174in HEK TLR-4 cells. (Figure 1.16A) Cellular uptake images of FITC-PEG5kSUA8C174at differentconcentrations in HEK TLR-4 cells for 2.5 h. (Figure 1.16B) Cell images of HEK TLR- 4 cells incubated with 100 pg / mL FITC-LPS and 0, 100, 200 and 500 pg / mL Rh- PEG5kSUA8C174for 2 h.

[0034] Figure 1.17 presents cellular uptake of PEG5kSUA8C174 in RAW 264.7 cells. (Figure 1.17A) Cellular uptake images of FITC-PEG5kSUA8C174 at different concentrations in RAW 264.7 cells for 2.5 h. (Figure 1 .17B) Cell images of RAW 264.7 cells incubated with 100 pg / mL FITC-LPS and 0, 100, 200 and 500 pg / mL Rh- PEG5kSUA8C174for 2 h.

[0035] Figure 1.18 presents images of cell adherence to TD functionalized PEGA-resins. HEK-TLR4, HEK-TLR2 and WT HEK attached to PEGA-SUA8C174, PEGA-SUA4C174, PEGA-SUA4OAC4 and PEGA-C174 resins.

[0036] Figure 1.19 presents hemolytic toxicity of TD examples. TDs include PEG5kSUA8OAc4, PEG5kSUA8C44, PEG5kSUA8PPA4, PEG5kSUA8PBA4,PEG5kSUAsC94, PEG5kSUA8C174, PEG5kSAsC174, PEG5kOA8C174, PEG5kPA8C174, PEG5kSUA8C124, PEG5kSUA8C144, PEG5kSUA8C164, PEG5kSUA8C184,PEG5kSUA8CHO4, PEG5kSUA8VE4, PEG5kSUA8Rf4, PEG5kSUA8CA4, PEG5kSUA8Flu4, PEG20kSUA8C174, PEG2kSUA8C174, GPC8SUA8C174, PEG5kSUA8- L-C174.

[0037] Figure 1.19 presents IVIS image of i.v. LPS-induced bioluminescence in HLL mice. Figure 1.19A IVIS images of 0.5 mg / kg LPS induced bioluminescence treated by i.v. 25, 50, and 250 mg / kg PEG5kSUAsC174 in HLL mice at different time points. Figure 1.19B Bioluminescence signal of LPS and LPS+ PEG5kSUAsC174 treatment groups at different time points and their AUC. Figure 1.19C Temperature and body weight change of the mice in control, LPS and LPS+ PEG5kSUA8C174 treatment groups at different time points. Figure 1.19D Serum proinflam matory cytokines TNF-a and IL-6 levels of control, LPS and LPS+ PEG5kSUAsC174 treatment groups at 3 h after injection.

[0038] Figure 1 .20 presents PK and biodistribution of Rh-PEG5kSUA8C174. FIG.1.20A PK of Rh-PEG5kSUA8C174 via i.v. and i.p. injection in the mice without or with 4-dose of PEG5kSUA8C174 pretreatment. FIG. 1.20B Ex vivo images and biodistribution of Rh-PEG5kSUAsC174 via i.v. or i.p. injection in the mice without or with 4-dose of PEG5kSUA8C174 pretreatment.

[0039] Figure 1.21 presents in vitro characterization of GM nanoformulations. (Figure 1.21 A) Ischemic GM-TD nanoformulation via charge and hydrophobicinteractions. (Figure 1.21 B) Particle size, zeta potential of blank TD and GM encapsulated TD at neutral and acidic pH. (Figure 1.21C)1H NMR of free GM, GM- PEG5kSUA8C174, GM-PEG5kSA8C174, GM-PEG5kOA8C174, and GM-PEG5kPA8C174in D2O. (Figure 1.21 D) GM release profiles of free GM, GM-PEG5kSUA8C174, GM- PEG5kSA8C174, GM-PEG5kOA8C174, and GM-PEG5kPA8C174at neutral and acidic pH.

[0040] Figure 1.22 presents cell viability and cellular uptake of GM nanoformulations. (Figure 1.22A) Cell viability of 293T, THP-1 and RAW 264.7 cells incubated with free GM, GM-PEG5kSUA8C174, GM-PEG5kSA8C174, GM- PEG5kOA8C174, and GM-PEG5kPA8C174at different concentrations for 72 h. (Figure 1.22B) Cell viability of 293T, THP-1 and RAW 264.7 cells incubated with PEG5kSUA8C174, PEG5kSA8C174, PEG5kOA8C174, and PEG5kPA8C174at different concentrations for 72 h. (Figure 1.22C) RAW 264.7 cellular uptake of GM-FITC- PEG5kSUA8C174, GM-FITC-PEG5kSA8C174, GM-FITC-PEG5kOA8C174, and GM- FITC-PEG5kPA8C174for 4 h. (Figure 1.22D) Hemolytic toxicity of free GM, GM- PEG5kSUA8C174, GM-PEG5kSA8C174, GM-PEG5kOA8C174, and GM-PEG5kPA8C174with different concentrations at 0.5 h, 4 h and overnight incubation.

[0041] Figure 1.23 presents MIC and intracellular bacteria inhibition of GM nanoformulations. (Figure 1.23A) MIC of free GM, GM-PEG5kSUA8C174, GM- PEG5kSA8C174, GM-PEG5kOA8C174, GM-PEG5kPA8C174, PEG5kSUA8C174, PEG5kSA8C174, PEG5kOA8C174, and PEG5kPA8C174incubated with gram-positive and gram-negative bacteria at different concentrations for overnight. (Figure 1 .23B) Intracellular bacteria CFU (20 pL) of infected RAW 264.7 cells treated by free GM, GM-PEG5kSUA8C174, GM-PEG5kSA8C174, GM-PEG5kOA8C174, GM-PEG5kPA8C174.

[0042] Figure 1.24 presents images of intracellular infected E. Coli treated by GM and nanoformulations. Intracellular infected E.Coli inhibition of free GM, GM- PEG5kSUA8C174, GM-PEG5kSA8C174, GM-PEG5kOA8C174, and GM-PEG5kPA8C174. PI stain for dead E. Coli, SYTO 9 stain for live E. Coli.

[0043] Figure 1.25 presents images of intracellular infected Pseudo, aeruginosa treated by GM and nanoformulations. Intracellular infected Pseudo, aeruginosa inhibition of free GM, GM-PEG5kSUA8C174, GM-PEG5kSA8C174, GM- PEG5kOA8C174, and GM-PEG5kPA8C174. PI stain for dead Pseudo, aeruginosa, SYTO 9 stain for live Pseudo, aeruginosa.

[0044] Figure 1.26 presents images of intracellular infected Lis. monocytogenes treated by GM and nanoformulations. Intracellular infected Lis. monocytogenes inhibition of free GM, GM-PEG5kSUA8C174, GM-PEG5kSAsC174, GM- PEG5kOAsC174, and GM-PEG5kPAsC174. PI stain for dead Lis. monocytogenes, SYTO 9 stain for live Lis. Monocytogenes

[0045] Figure 1.27 presents LPS-induced proinflammatory cytokines TNF-a and IL-6 levels produced by RAW 264.7 cells with GM, GM nanoformulation and blank TD treatment. (Figure 1.27A) 50 ng / mL LPS-induced TNF-a and IL-6 levels produced by RAW 264.7 cells with overnight treatment of free GM, GM-PEG5kSUA8C174, GM- PEG5kSA8C174, GM-PEG5kOA8C174, and GM-PEG5kPA8C174. (Figure 1.27B) 50 ng / mL LPS-induced TNF-a and IL-6 levels produced by RAW 264.7 cells with overnight treatment of GM-PEG5kSUA4C174, GM-PEG5kSA4C174, GM- PEG5kOA4C174, and GM-PEG5kPA4C174. (Figure 1.27C) 50 ng / mL LPS-induced TNF- a and IL-6 levels produced by RAW 264.7 cells with overnight treatment of PEG5kSUA8C174, PEG5kSA8C174, PEG5kOA8C174, PEG5kPA8C174, and PEG5kArg8C174. (Figure 1.27D) 50 ng / mL LPS-induced TNF-a and IL-6 levels produced by RAW 264.7 cells with overnight treatment of PEG5kSUA4C174, PEG5kSA4C174, PEG5kOA4C174, PEG5kPA4C174, and PEG5kArg4C174. (Figure 1 .27E) Supernatant of 50 ng / mL LPS-induced TNF-a and IL-6 levels produced by RAW 264.7 cells mixed with different concentrations of PEG5kSUAsC174, PEG5kSAsC174, PEG5kOA8C174, PEG5kPA8C174, PEG5kArg4C174 and PEG5kArg8C174.

[0046] Figure 1 .28 presents gram-negative and gram-positive bacteria induced proinflammatory cytokines TNF-a produced by RAW 264.7 and THP-1 cells with GM, GM nanoformulation and blank TD treatment. (Figure 1.28A) Gram-negative and gram-positive bacteria induced proinflammatory cytokines TNF-a produced by RAW 264.7 cells with 4 h treatment of free GM, GM-PEG5kSUA8C174and PEG5kSUA8C174and OD of bacteria growth overnight. (Figure 1 .28B) Gram-negative and gram-positive bacteria induced proinflammatory cytokines TNF-a produced by THP-1 cells with 4 h treatment of free GM, GM-PEG5kSUA8C174 and PEG5kSUA8C174 and OD of bacteria growth overnight. (Figure 1.28C) Gram-negative and gram-positive bacteria induced proinflammatory cytokines TNF-a produced by BMDM cells with 4 h treatment of free GM, GM-PEG5kSUA8C174 and PEG5kSUA8C174 and OD of bacteria growth overnight. (Figure 1.28D) Gram-negative and gram-positive bacteria induced TLR4 pathway activation by HEK TLR-4 cells with 4 h treatment of free GM, GM-PEG5kSUA8C174and PEG5kSUA8C174. (Figure 1.28E) Gram-negative and gram-positive bacteria induced TLR2 pathway activation by HEK TLR-2 cells with 4 h treatment of free GM, GM-PEG5kSUA8C174and PEG5kSUA8C174.

[0047] Figure 1 .29 presents gram-negative and gram-positive bacteria induced proinflammatory cytokines TNF-o produced by RAW 264.7 and THP-1 cells with PMB, DAP, PMB or DAP nanoformulation and blank TD treatment. (Figure 1.29A) Gramnegative and gram-positive bacteria induced proinflammatory cytokines TNF-a produced by RAW 264.7 cells with 4 h treatment of free PMB, DAP, PMB- PEG5kSUA8C174, DAP-PEG5kSUA8C174and PEG5kSUA8C174and OD of bacteria growth overnight. (Figure 1.29B) Gram-negative and gram-positive bacteria induced proinflammatory cytokines TNF-a produced by THP-1 cells with 4 h treatment of free PMB, DAP, PMB-PEG5kSUA8C174, DAP-PEG5kSUA8C174and PEG5kSUA8C174and OD of bacteria growth overnight. (Figure 1.29C) Gram-negative and gram-positive bacteria induced proinflammatory cytokines TNF-a produced by BMDM cells with 4 h treatment of free PMB, DAP, PMB-PEG5kSUA8C174, DAP-PEG5kSUA8C174and PEG5kSUA8C174.

[0048] Figure 1 .30 presents gram-negative and gram-positive bacteria induced TLR pathway activation in HEK TLR cells with PMB, DAP, PMB or DAP nanoformulation and blank TD treatment. (Figure 1.30A) Gram-negative and grampositive bacteria induced TLR2 pathway activation by HEK TLR-2 cells with 4 h treatment of free PMB, DAP, PMB-PEG5kSUA8C174, DAP-PEG5kSUA8C174and PEG5kSUA8C174. (Figure 1.30B) Gram-negative and gram-positive bacteria induced TLR4 pathway activation by HEK TLR-4 cells with 4 h treatment of free PMB, DAP, PMB-PEG5kSUA8C174, DAP-PEG5kSUA8C174and PEG5kSUA8C174.

[0049] Figure 1 .31 presents TLR agonist properties of TDs. (Figure 1 .31 A) TNF- a production of RAW 264.7 and THP-1 cells incubated with PEG5kSUA8OAc4, PEG5kSUA8C44, PEG5kSUA8PPA4, PEG5kSUA8PBA4, PEG5kSUA8C94, PEG5kSUA8C174. (Figure 1.31 B, Figure 1 .31 C) TLR activation of HEK TLR-2, HEK TLR-4 and THP-1 blue cells incubated with PEG5kSUA8OAc4, PEG5kSUA8C44, PEG5kSUA8PPA4, PEG5kSUA8PBA4, PEG5kSUA8C94, PEG5kSUA8C174. (Figure 1.31 D) TNF-a production of RAW 264.7 induced by PEG5kSUA8C44 and treated with PEG5kSUA8C174, PEG5kSUA8CHO4and PEG5kSUA8C144.

[0050] Figure 1 .32 presents TLR agonist properties of TDs. (Figure 1 .32A) TNF- a production of RAW 264.7 and THP-1 cells incubated with PEG2kSUA8C176 andPEG5kSUA6C176. (Figure 1.32B) TLR activation of HEK TLR-2 and HEK TLR-4 blue cells incubated with PEG2kSUA6C176and PEG5kSUA6C176. (Figure 1.32C) TNF-a production of whole human blood incubated with PEG2kSUA6C176 and PEG5kSUA6C176and treated with PEG5kSUA8C174.

[0051] Figure 1 .33 presents TLR agonist properties of TDs. (Figure 1 .33A) TNF- a production of RAW 264.7 and THP-1 cells incubated with PEG5kPA2C9e and PEG5kPA2C12e, and inhibition of LPS-induced TNF-a production by PEG5kPA2C98and PEG5kPA2C126. (Figure 1.33B) TLR activation of HEK TLR-2 and HEK TLR-4 blue cells incubated with PEG2kSUA8C176 and PEG5kSUA6C176 and inhibition of LPS- activated TLR pathway by PEG5kPA2C98and PEG5kPA2C12e.

[0052] Figure 1.34 presents oligomer immunomodulation properties. (Figure 1.34A) Inhibition of LPS-induced RAW 264.7 TNF-a production by SUA8OAc4, SUA8C44, SUA8PPA4, SUA8PBA4, SUA8C94, SUA8C174. (Figure 1.34B) Inhibition of LPS-activated TLR4 in HEK TLR-4 cells by SUA8OAc4, SUAsC44, SUA8PPA4, SUA8PBA4, SUA8C94, SUA8C174. (Figure 1.34C) Inhibition of LPS-activated TLR2 in HEK TLR-2 cells by SUA8OAc4, SUA8C44, SUA8PPA4, SUA8PBA4, SUA8C94, SUA8C174.

[0053] Figure 1 .35 presents inhibition of TLR 7 / 9 / 3 agonist I MQ, CPG, Poly (l:C) by TDs. (Figure 1.35A) IMQ induced TNF-a production in RAW 264.7 and THP-1 cells incubated with different TDs. (Figure 1.35B) CPG or Poly (l:C) induced TNF-a production in RAW 264.7 and THP-1 cells incubated with different TDs.

[0054] Figure 1.36 presents inhibition of TNF-a induced TNF-a production by TDs. (Figure 1.36A) TNF-a induced TNF-a production in RAW 264.7 and THP-1 cells incubated with different TDs. (Figure 1 .36B) TNF-a, IL-1 and Poly(l:C) induced TLR activation in THP-1 blue cells incubated with different TDs.

[0055] Figure 1.37 presents inhibition of TNF-a or Poly(l:C) activated TLR pathway by TDs in HEK TLR-3 cells. (Figure 1 .37A) TNF-a activated TLR pathway in HEK TLR-3 cells incubated with different TDs. (Figure 1.37B) Poly(l:C) activated TLR pathway in HEK TLR-3 cells incubated with different TDs.

[0056] Figure 2.1 presents the characterization of the disclosed telodendrimer nanoparticles. (Figure 2.1 A) Encapsulation of polymyxin B with Td through both electrostatic and hydrophobic interactions to form nano-PMB. (Figure 2.1 B) Particle size and (Figure 2.1 C) zeta potential of the nano-PMB at pH 7.4 (phosphate buffer). (Figure 2.1 D) In vitro PMB release from nano-PMB in PBS at 37°C. Agarose GelElectrophoresis to determine the drug loading efficiency of the individual nano-PMB after overnight incubation with (Figure 2.1 E) Fetal bovine serum (FBS) or (Figure 2.1 F) Bovine serum albumin (BSA). Note: TD(SUA) has a detailed structure of PEG5kSUA8C174 : SUA refers to succinic acid.

[0057] Figure 2.2 presents Nano-PMB reduces the toxicity of PMB without compromising antibacterial efficacy. Cell viability assay with (Figure 2.2A) PMA- differentiated THP-1 cell line (human macrophages), (Figure 2.2B) Raw 264.7 cell line (mouse macrophages), (Figure 2.2C) 293t cell line, and (Figure 2.2D) Hemolysis study of the individual nano-PMB and free PMB. Antibacterial efficacy of the individual nano- PMB. Dose-dependent effect of each drug on (Figure 2.2E) E. coli K12 (top) and P. aeruginosa (bottom), (Figure 2.2F) MIC90 values of the treated samples. (Figure 2.2G) Confocal and SEM images of cells. Note: TD(SUA) has a detail structure of PEG5kSUA8C174 : SUA refers to succinic acid.

[0058] Figure 2.3 presents cell Cytotoxicity of nano-PMB. Cell viability assay with (Figure 2.3A) PMA-differentiated ATCC THP-1 cell line (human macrophages, 50k cel Is / wel I ) , (Figure 2.3B) Raw 264.7 cell line (mouse macrophages, 15k cells / well), (Figure 2.3C) Bone-marrow-derived macrophages (20k cells / well). LDH release assay (Figure 2.3D) PMA-differentiated ATCC THP-1 cell line (human macrophages, 50k cells / well), (E) Raw 264.7 cell line (mouse macrophages, 15k cells / well), (Figure 2.3F) Bone-marrow-derived macrophages (20k cells / well). Note: TD in this Figure 2-refers to TD(SUA) with a detail structure of PEG5kSUAsC174

[0059] Figure 2.4 presents an inti-inflammatory activity of the nanoformulation through TLR4 and TLR2 inhibition. (Figure 2.4A) Anti-LPS activity of the free PMB, individual nano-PMB, and empty nanoparticles after overnight stimulation of Raw 264.7 cells with E. coli LPS (10ng / mL) and drug (0.5 ug / mL) or NP (5 ug / mL) and analyzed for mTNF-a and mlL-6. (Figure 2.4B) Anti-LPS and (Figure 2.4C) Anti-LTA activity of the free PMB, TD(SUA):PMB, TD(SUA), and LPS:RS after overnight stimulation of PMA-differentiated THP-1 cells with LPS (50 ng / mL) and drug (4 ug / mL) or NP (40 ug / mL) and analyzed for hTNF-a, hlL-6, and hlL1-b. (Figure 2.4D) SEAP production from HEK-Blue hTLR4 (left) and Dual HEK hTLR2 (right) reporter cells incubated for 24h with LPS as a mixture with free PMB, TD(SUA):PMB, TD(SUA), and LPS: RS. All the data presented were triplicated. Statistical significance was done by multiple comparison tests following one-way ANOVA (****p<0.0001 ; ns, no significantdifference). Note: TD(SLIA) has a detailed structure of PEG5kSUAsC174 : SUA refers to succinic acid.

[0060] Figure 2.5 presents an anti-inflammatory activity of the nanoformulation through other TLR and non-TLR inhibition. (Figure 2.5A) Anti-PAMP (Gram-negative bacteria) activity of the free PMB, TD(SUA):PMB and TD(SLIA) after overnight stimulation of THP-1 cells with E. col i and P. aeruginosa PAMP (50 ug / mL) and drug (4 ug / mL) or NP (40 ug / mL) and analyzed for hTNF-a (left two), and hlL-6 (right two) by ELISA. (Figure 2.5B) Anti-PAMP (Gram-positive bacteria) activity of the free PMB, TD(SUA):PMB and TD(SUA) after overnight stimulation of THP-1 cells with S. aureus PAMP (50 ug / mL) and drug (4 ug / mL) or NP (40 ug / mL) and analyzed for hTNF-a (left), and hlL-6 (right) by ELISA. (Figure 2.5C) Anti-TNF-a (Non-TLR Ligand) activity after overnight stimulation of THP-1 cells with TNF-a (100 pg / mL) and drug (4 ug / mL), NP (40 ug / mL) and analyzed for hTNF-a by ELISA. (Figure 2.5D) SEAP production from HEK-Blue hTLR4 reporter cells incubated for 24h with hTNF-a as a mixture with TD(SUA):PMB and TD(SUA) by ELISA. (Figure 2.5E) Anti-DAMP activity of the TD(SUA):PMB and TD(SUA) after overnight stimulation of Raw 264.7 cells with mPANCO2 DAMP (250 ug / mL) and drug (4 ug / mL) or NP (40 ug / mL) and analyzed for mTNF-a. All the data presented were triplicated. Statistical significance was done by multiple comparison tests following one-way ANOVA (****p<0.0001 ; ns, no significant difference). Note: TD in this Figure 2-refers to TD(SUA) with a detailed structure of PEG5kSUA8C174

[0061] Figure 2.6 presents attenuation of LPS-induced NF-kB Activation in HLL mice. In vivo anti-inflammation effect of telodendrimer (TD) nanoformulated polymyxin B (PMB) was investigated using HLL NF-kB reporter mice. LPS (E. coli, 0.2 mg / kg) was mixed with different PMB nanoformulations in tubes and immediately i.v. injected through tail vein. TD only and LPS only serve as control groups. (Figure 2.6A) Real time in vivo bioluminescence imaging and Figure 2.6 (B) the quantitative bioluminescence analysis of abdominal area at time points of 0, 2, 4, 6, 24 h after injection. (Figure 2.6C) Cytokine analysis (mlL-6) after 3hr of infection compared with treated and non-treated groups. (n=3, mean + SD). Note: TD in this Figure 2-refers to TD(SUA) with a detail structure of PEG5kSUA8C174 : (SUA refers to succinic acid).

[0062] Figure 2.7 presents an anti-inflammatory activity of the nanoformulation. Anti-LPS activity of the free PMB, TD(SUA):PMB and TD(SUA) alone after 2hr stimulation of LPS (50 ng / mL) in (Figure 2.7A) Healthy human whole blood and (Figure2.7B) Sepsis patient whole blood analyzed for hTNF-a by ELISA. All the data presented were triplicated. Statistical significance was done by multiple comparison tests following one-way ANOVA (****p<0.0001 ; ns, no significant difference). Note: TD in this Figure 2-refers to TD(SLIA) with a detailed structure of PEG5kSUA8C174 : (SUA refers to succinic acid).

[0063] Figure 2.8 presents inhibition of NF-kB / IKBa Phosphorylation against LPS (Gram-negative bacterial ligands). (FIG. 2.8A) Inhibition of TLR-LPS interaction by the TD nanoparticle. (FIG. 2.8B) Raw 264.7 cells were stimulated with LPS 1 ug / mL for 6hr with and without PMB (20ug / mL), TD:PMB (20ug / mL), TD only (250ug / mL), and LPS:RS (20ug / mL); then cytokine analysis was done by ELISA. (FIG. 2.8C) Representative western blot images of three independent experiments where protein expression of both NFkB and IKBa and phosphorylation of these two were measured. Quantification of the (FIG. 2.8D) phospho NFkB p65 / p65 and (FIG. 2.8E) phospho IKBa / IKBa protein expression. Imaged software was used for measuring the protein band’s optical density. B-actin was used as a loading control. Statistical significance was done by multiple comparison tests following one-way ANOVA (****p<0.0001 ; ns, no significant difference). Note: TD in this Figure 2-refers to TD(SUA) with a detail structure of PEG5kSUA8C174 : (SUA refers to succinic acid).

[0064] Figure 2.9 presents the inhibition of NF-kB / IKBa Phosphorylation against LTA (Gram-positive bacterial ligands). (FIG. 2.9A) Inhibition of TLR-LTA interaction by the TD nanoparticle. (Figure 2.9B) Raw 264.7 cells were stimulated with LTA 50ug / mL for6hrwith and without PMB (20ug / mL), TD:PMB (20ug / mL), and TD only (250ug / mL), then cytokine analysis was done by ELISA. (Figure 2.9C) Representative western blot images of three independent experiments where protein expression of both NFkB and IKBa and phosphorylation of these two were measured. Quantification of the (Figure 2.9D) phosphor NFkB p65 / p65 and € phosphor IKBa / IKBa protein expression. (Figure 2.9E) phospho IKBa / IKBa protein expression. Imaged software was used for measuring the protein band’s optical density. B-actin was used as a loading control. Statistical significance was done by multiple comparison tests following one-way ANOVA (****p<0.0001 ; ns, no significant difference). Note: TD in this Figure 2-refers to TD(SUA) with a detail structure of PEG5kSUA8C174 : (SUA refers to succinic acid).

[0065] Figure 2.10 presents the inhibition of NF-kB / IKBa Phosphorylation against TNFa (non-TLR ligands). (Figure 2.10A) Inhibition of TNFaR-TNFa interaction by the TD nanoparticle. (Figure 2.10B) Raw 264.7 cells were stimulated with mTNFa350ug / mL for 6hr with and without PMB (20ug / mL), TD:PMB (20ug / ml_), and TD only (250ug / mL), then cytokine analysis was done by ELISA. (Figure 2.1 OC) Representative western blot images of three independent experiments where protein expression of both NFkB and IKBa and phosphorylation of these two were measured. Quantification of the (Figure 2.10D) phospho NFkB p65 / p65 and (Figure 2.10E) phospho IKBa / IKBa protein expression. Imaged software was used for measuring the protein band’s optical density. B-actin was used as a loading control. Statistical significance was done by multiple comparison tests following one-way ANOVA (****p<0.0001 ; ns, no significant difference). Note: TD in this Figure 2-refers to TD(SUA) with a detail structure of PEG5kSUA8C174 : (SUA refers to succinic acid).

[0066] Figure 2.11 presents the inhibition of hyper-inflammation by TD nanoparticle after antibiotic (imipenem) treatment. We treated E. coli DH5a for 24hr with antibiotic like imipenem (I Ml) and then collected those cell lysate (PAMPs). Then based on protein concentration we added 50ug / mL of this EC PAMPs in Raw 264.7 cells with and without the pretreatment of TD nanoparticle. Then incubated for 4hr (Figure 2.11 A) Imipenem 5ug / mL (Figure 2.11 B) Imipenem / cilastatin 5 / 5 ug / mL and 24hr (Figure 2.11C) Imipenem 5ug / mL (Figure 2.11 D) Imipenem / cilastatin 5 / 5 ug / mL. Finally, the supernatants were used for cytokine analysis. Statistical significance was done by multiple comparison test following one-way ANOVA (****p<0.0001 ;ns, no significant difference). Note: TD in this Figure 2-refers to TD(SUA) with a detail structure of PEG5kSUA8C174 : (SUA refers to succinic acid).

[0067] Figure 2.12 presents the Maximum Tolerated Dose (MTD) Study in C57BL / 6J Mice. (Figure 2.12A) Drugs were intravenously injected into C57BL / 6J mice (8-12 weeks) through a tail vein for seven days. Real-time in vivo bioluminescence imaging and (Figure 2.12B) Drug doses were summarized and (Figure 2.12C) Survival curve showed the benefit of TD:PMB. (Figure 2.12D) Mice blood 7 day treatment. (FIG. 2.12E) Mice 7 blood day treatment. Then the mice were sacrificed to collect the blood and BUN (kidney toxicity) and ALT (hepatotoxicity) levels were measured using a commercial kit. Note: TD in this Figure 2-refers to TD(SUA) with a detailed structure of PEG5kSUAsC174 : (SUA refers to succinic acid).

[0068] Figure 2.13 presents the photomicrographs of hematoxylin and eosin (H&E)-stained heart, liver, and kidney sections of C57BL / 6 mice after 7 days of treatments with a single IV injection (per day) of 5 mg / kg PMB, TD(SUA):PMBequivalent to 5 mg / kg and 7.5 mg / kg PMB, 250 mg / kg of TD(SUA) only. The images are representative of specimens from 3 mice per group. Scale bars: 20 pm.

[0069] Figure 2.14A and Figure 2.14B present E. ColiDH5a induced sepsis (Bacteremia) study in C57BL / 6J mice (4thbatch).

[0070] Figure 2.15 presents the results for E. Coli DH5a induced Sepsis (Bacteremia) treatment in C57BL / 6J mice (4thbatch) as illustrated in Figure 2.14. (Figure 2.15A): Mice were infected with E. coli DH5a isolate (5.5*10A8 cells / mice) via intraperitoneal (i.p.) injection and then treated with PMB (5 mg / kg), TD(SUA):PMB (5 mg / kg) or TD(SUA) (250 mg / kg) post 2hr infection. (Figure 2.15B) Survival for the different treatment groups was monitored and analyzed (n = 6-7). No additional mortality was observed after 168 hr. (Figure 2.15A) Their body temperature and (Figure 2.15B) body weight change were monitored for 7 days. (Figure 2.15C) The blood plasma (supernatants) was collected after 4hr and 24hr post-infection for cytokine analysis. Statistical significance was done by multiple comparison tests following one-way ANOVA (****p<0.0001 ; ns, no significant difference).

[0071] Figure 2.16. presents E. Coli DH5a induced Sepsis (Bacteremia) Study in C57BL / 6J mice treated with different formulation of PMB and IMI / IC antibiotics.DEFINITIONS

[0072] As used in the present specification, the following words and phrases are generally intended to have the meanings as set forth below, except to the extent that the context in which they are used indicates otherwise.

[0073] As used herein, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, references to “a compound” include the use of one or more compound(s).

[0074] As used herein the terms “about,” “approximately,” and the like, when used in connection with a numerical variable, generally refers to the value of the variable and to all values of the variable that are within the experimental error (e.g., within the 95% confidence interval [Cl 95%] for the mean) or within ±10% of the indicated value, whichever is greater.

[0075] As used herein the terms “drug,” “drug substance,” “active pharmaceutical ingredient,” and the like, refer to a compound that may be used for treating a subject in need of treatment.

[0076] As used herein the term “excipient” or “adjuvant” refers to any inert substance.

[0077] As used herein the terms “drug product,” “pharmaceutical dosage form,” “dosage form,” “final dosage form” and the like, refer to a pharmaceutical composition that is administered to a subject in need of treatment and generally may be in the form of inhalers, tablets, capsules, sachets containing powder or granules, liquid solutions or suspensions, patches, and the like.

[0078] As used herein the term “pharmaceutically acceptable” substances refers to those substances, which are within the scope of sound medical judgment suitable for use in contact with the tissues of subjects without undue toxicity, irritation, allergic response, and the like, and effective for their intended use.

[0079] As used herein the term “pharmaceutical composition” refers to the combination of one or more drug substances, one or more excipients, and one or more pharmaceutically acceptable vehicles with which the one or more drugs is administered to a subject.

[0080] As used herein, the term "telodendrimer" refers to a dendrimer containing a hydrophilic PEG segment and one or more chemical moieties covalently bonded to one or more end groups of the dendrimer. These moieties can include, but are not limited to, hydrophobic groups, hydrophilic groups, amphiphilic compounds, and drugs. Different moieties may be selectively installed at desired end groups using orthogonal protecting group strategies.

[0081] As used herein, the terms "dendrimer" and "dendritic polymer" refer to branched polymers containing a focal point, a plurality of branched monomer units, and a plurality of end groups. The monomers are linked together to form arms (or "dendrons") extending from the focal point and terminating at the end groups. The focal point of the dendritic polymer can be attached to other segments of the telodendrimers, and the end groups may be further functionalized with additional chemical moieties.

[0082] As used herein, the terms "monomer" and "monomer unit" refer to repeating units that make up the dendrons of the dendritic polymers of the invention. The monomers may be AB2-type monomers, where A and B are two different functional groups capable of reacting together such that the resulting polymer chain has a branch point where an A-B bond is formed. Exemplary monomers include a diamino carboxylic acid, a dihydroxy carboxylic acid and a hydroxyl amino carboxylicacid. Examples of diamino carboxylic acid groups of the present invention include, but are not limited to, 2,3-diamino propanoic acid, 2,4-diaminobutanoic acid, 2,5- diaminopentanoic acid (ornithine), 2,6-diaminohexanoic acid (lysine), (2-Aminoethyl)- cysteine, 3-amino-2-aminomethyl propanoic acid, 3-amino-2-aminomethyl-2-methyl propanoic acid, 4-amino-2-(2-aminoethyl)butyric acid and 5-amino-2-(3- aminopropyl)pentanoic acid. Examples of dihydroxy carboxylic acid groups of the present invention include, but are not limited to, glyceric acid, 2,4-dihydroxybutyric acid, and 2,2-bis(hydroxymethyl)propionic acid. Examples of hydroxyl amino carboxylic acids include, but are not limited to, serine and homoserine. One of skill in the art will appreciate that other monomer units are useful in the present invention.

[0083] As used herein, the term "amino acid" refers to a carboxylic acid bearing an amine functional group. Amino acids include the diamino carboxylic acids described above. Amino acids include naturally occurring .alpha. -amino acids, wherein the amine is bound to the carbon adjacent to the carbonyl carbon of the carboxylic acid. Examples of naturally occurring .alpha. -amino acids include, but are not limited to, L- aspartic acid, L-glutamic acid, L-histidine, L-lysine, and L-arginine. Amino acids may also include the D-enantiomers of naturally occurring .alpha. -amino acids, as well as .beta. -amino acids and other non-naturally occurring amino acids.

[0084] As used herein, the term "lipid" refers to lipid molecules that can include fats, waxes, steroids, cholesterol, fat-soluble vitamins, monoglycerides, diglycerides, triglycerides, phospholipids, sphingolipids, glycolipids, cationic or anionic lipids, derivatized lipids, and the like, as described in detail below. Lipids can form micelles, monolayers, and bilayer membranes.

[0085] As used herein, the term "hydrophobic group" refers to a chemical moiety that is water-insoluble or repelled by water. Examples of hydrophobic groups include, but are not limited to, long-chain alkanes and fatty acids, fluorocarbons, silicones, certain steroids such as cholesterol, and many polymers including, for example, polystyrene and polyisoprene.

[0086] As used herein, the term "hydrophilic group" refers to a chemical moiety that is water-soluble or attracted to water. Examples of hydrophilic groups include, but are not limited to, alcohols, short-chain carboxylic acids, quaternary amines, sulfonates, phosphates, sugars, and certain polymers such as polyethylene glycol) (PEG).

[0087] As used herein, the term "amphiphilic compound" refers to a compound having both hydrophobic portions and hydrophilic portions. For example, the amphiphilic compounds of the present invention can have one hydrophilic face of the compound and one hydrophobic face of the compound. Amphiphilic compounds useful in the present invention include, but are not limited to, cholic acid and cholic acid analogs and derivatives, and cholesterol formate.

[0088] As used herein the term nanodrug refers to the use of nano-construct as a therapeutic reagent directly. Nanodrug exerts bioactivity as the individual assembly unit of nanoparticles. Nanodrugs exhibit bioactivity through interaction with specific molecules, receptors, pathway and cell organelles to intervene cellular function or systemic changes in pathophysiological process. Nanodrug could also serve as nanocarrier for the encapsulation and delivery of other therapeutics, e.g. small molecular drug, gene molecules, peptide / proteins.

[0089] As used herein the term nanocarriers refers to particles having at least one of their dimensions within 1-100 nm range and are engaged in transporting another substance, such as, a drug, gene, protein, and the like. Examples of different types of nanocarriers are polymeric or solid lipid nanoparticles, lipid or albumin nanocapsules, liposomes and micelles, dendrimers, telodendrimers, nanovesicles, nanogels, nano-emulsions, and nanosuspensions.

[0090] As used herein the term nanotherapeutics refers to the use of nanotechnology, i.e. nano-size carrier called nanocarriers, to improve drug delivery to specifically targeted organs. Nanotherapeutics utilize various nanocarrier systems that have been developed for delivery of different therapeutic drugs via different routes of administration. Nanotherapeutics have been employed to improve the efficacy, safety, physicochemical properties, and pharmacokinetic / pharmacodynamic profile of pharmaceutical substances. In particular, functionalized nanotherapeutics can offer enhanced bioavailability of orally taken drugs, prolonged half-life of injected drugs (by reducing immunogenicity), and targeted delivery to specific tissues.

[0091] As used herein the phrase immune modulating activity refers to the alteration of immune cell response in infection and inflammation. As such, a substance that stimulates or suppresses the immune system, may help the body fight cancer, infection, or other diseases. Immune modulating activity can be exhibited by immunomodulating agents, such as compounds and composition disclosed herein ,monoclonal antibodies, cytokines, and vaccines, all of which affect specific parts of the immune system.

[0092] As used herein the phrase Damage-associated molecular patterns (DAMPs) refers to endogenous danger molecules that are released from damaged or dying cells and activate the innate immune system by interacting with pattern recognition receptors (PRRs).

[0093] As used herein the phrase Pathogen-associated molecular patterns (PAMPs) refers to pathogen-specific carbohydrates and lipoproteins or nucleic acids expressed as part of their life cycle (i.e. , bacterial DNA as unmethylated repeats of dinucleotide CpG, double-stranded [ds] or single-stranded [ss] RNA). These pathogens are recognized by TLRs and activate cytokines including IL-1 p and TNF-a.

[0094] As used herein the term Toll-like receptor (TLRs) refers to type 1 transmembrane proteins with an extracellular domain for ligand binding composed of leucine rich repeats and a cytoplasmic domain for intracellular signaling which is known as the Toll / IL-1 receptor (TIR) domain. TLRs recognize bacteria, viruses, fungi, protozoa, and endogenous ligands, such as heat shock proteins and fibrinogen.

[0095] As used herein the phrase Receptor-residing lipid raft refers to dynamic assemblies of proteins and lipids that harbor many receptors and regulatory molecules and so act as a platform for signal transduction. They float freely within the liquid- disordered bilayer of cellular membranes and can cluster to form larger ordered domains.

[0096] Before embodiments are further described, it is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0097] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0098] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.DESCRIPTION OF THE EMBODIMENTS

[0099] Embodiments disclosed herein provide a series of novel telodendrimer (TD) nanodrug (ND) (e.g. PEG5kCOOH8C174) having fine-tuned architectures for improving the systemic delivery of antibiotics. These nanocarriers are rationally designed with polyanionic charge and hydrophobic moieties for successful encapsulation of polycationic PMB through both electrostatic and hydrophobic interactions. In addition, these TD can inhibit immune cell inflammation stimulated by different immunogenic molecules, e.g. LPS, LTA, bacterial cell lysates and cytokines through pleiotropic mechanism. The hydrophobic core (0174) may boost the charge interactions between PMB and TD to increase drug-loading stability. Additionally, the polyethylene glycol (PEG)-based neutralized surface of these micelle nanostructures may lessen their non-specific bindings and tissue accumulation (e.g. kidney and brain) to reduce nephrotoxicity, and neurotoxicity. It may further decrease their fast uptake by macrophages which prolongs their availability in the bloodstream. The biocompatible nanoparticle formulation of PMB (nano-PMB) may reduce the side effects of the drug while still preserving its ability to fight infection and control systemic hyperinflammation during GN sepsis. In this regard, we used a multidisciplinary approach that includes synthesis, biochemical, molecular, and immunological assays in both in vitro and in vivo models. This allowed us to effectively administer the drug at an effective dose and control the cytokine storm and organ dysfunction.

[0100] Sepsis is a life-threatening health emergency affecting 1 .7 million adults in the US, causing 350,000 deaths annually. Over 60% of severe sepsis cases are associated with Gram-negative (GN) bacterial infection, where PAMPs (e.g., LPS) upon TLR-4 recognition stimulate extreme inflammatory responses (e.g., cytokinestorm) leading to sepsis. Polymyxin B (PMB) has high antibacterial and anti-LPS activity, but poor stability and dose-limiting cytotoxicity (e.g., nephrotoxicity) limit its clinical applications at the early stage of sepsis treatment. We have designed novel telodendrimer nanoparticles (TD NPs) with polyanionic and hydrophobic features to encapsulate amphiphilic PMB through both electrostatic and hydrophobic interactions. This nano-PMB improves stability and sustain PMB release in the bloodstream without compromising the drug efficacy, thus reducing side effects, and allowing its application as first-line therapy to control GN sepsis.

[0101] Synthesis and characterization of physical properties of TD nanoparticles and nano-PMB: A stepwise synthetic approach using peptide chemistry was applied to prepare a series of TD (including PEG5kCAsC174, PEG5kOAaC174, PEG5kPAsC174, PEG5kSAsC174) with different anionic charges and hydrophobic moieties. General procedure for TD synthesis is described briefly as following: the TDs are synthesized by solution-phase peptide chemistry starting from MeO-PEG- NH2 HCI or other primary amino group containing polymer or branched zwitterionic materials. N-terminal-protected lysine is used to synthesize the branched scaffold of TD. First, triethylamine (TEA, 1 equiv) is added to neutralize the hydrochloride on PEG togetherwith N,N’-Diisopropylcarbodiimide (DIC, 3 equiv) and N-hydroxybenzotriazole (HOBt, 3 equiv) added as coupling reagents in dimethylformamide (DMF) to catalyze amide bond formation for N-terminal-protected lysine coupling. The reaction completion is confirmed by the negative Kaiser test result. The ice-chilled ether is added to the reaction solution to precipitate PEGylated intermediates, and then washed by chilled ether for three times. Then Fmoc-protection groups are removed by the treatment with 20% 4-methyl-piperidine in DMF for 30 min. PEGylated intermediate is precipitated and washed with chilled ether for three times. The Boc-protection groups are removed by the treatment with DCM: TFA (1 :1 v / v) for 30 min. The hydrophobic moieties and negatively charged moieties with carboxylic acid group are conjugated on primary amino of lysine with DIC and HOBt as coupling reagents. The hydrophobic moieties and negatively charged moieties with acyl chloride or anhydride group are conjugated on primary amino of lysine with TEA as catalyst. PEGylated intermediate is precipitated and washed with chilled ether for three times. The precipitated TDs are dissolved in deionized water and purified further via dialysis against deionized water for 2 days, followed by lyophilization. Each telodendrimer (TD) contains a long polyethylene glycol (PEG) chain with eight short branches of negativecharge moieties (e.g., carboxylic acid) and four branches of hydrophobic moieties (e.g., heptadecanoic acid or C17). This structure can be easily manipulated by modifying the number of charged branches or altering the hydrophobic moieties. The addition of a branched layer is controlled by the following steps. Initially, all of the active amine sites in the polymer was conjugated by two distinctive protection groups, namely Fmoc and Boc, in a predefined ratio. Then deprotection of the specific Fmoc groups to expose free amine groups which then blocked by hydrophobic heptadecanoic acid. Lastly, the Boc group is specifically deprotected to expose free amine groups, on which different negatively charged groups was conjugated. A ninhydrin test is performed at each conjugation step to confirm the completion of the reactions. The resultant polymers (e.g. PEG5kNegsC174) is then pass through the filtration and dialysis process and are subjected to lyophilization. Finally, the lyophilized blank TD samples is stored at room temperature and characterized by DLS, NMR, and MALDI-TOF MS for determining their physical properties in terms of particle size, molecular structure, and molecular weight, respectively.

[0102] In vitro biological evaluation such as antibacterial activity, and cell cytotoxicity was performed in Raw 264.7 and THP-1 cells. Anti-inflammatory properties were evaluated against broad-spectrum septic molecules (e.g., PAMPs and DAMPs) using ELISA and RT-PCR assay. We also used HEK hTLR4 and hTLR2 cells following Quanti-Blue (QB) assay to analyze specific TLR inhibitions. Luciferase activity was studied after co-injection of LPS and the nano-PMB in NF-KB reporter mice. Western blot analysis will be conducted to determine the nanoformulation’s effect on NF-KB pathway activity. Further, we investigated the pharmacokinetic profile, blood chemistry, and organ histopathology in the mice model study.

[0103] Nano-PMB demonstrated a slower drug release profile at physiological pH, suggesting prolonged duration in the bloodstream. Nano-PMB also showed similar Minimum Inhibitory Concentration values and less cell cytotoxicity than free PMB. It was more efficient in controlling cytokine production against both GN and Gram-positive PAMPs. Interestingly, empty NPs exhibited anti-inflammatory properties in a dose-dependent manner and with a structure-property relationship. Quanti-Blue assay confirmed that nanoformulations can control NF-KB and AP-1 pathway activity. Reduced bioluminescent signals from LPS-challenged NF-KB reporter mice treated with nanoformulations confirmed attenuated LPS-induced NF- KB activation.

[0104] Nano-PMB’s immune-modulatory effects is tested in endotoxemia and CLP mouse models to assess its potential for treating GN sepsis in humans. This unique nanoparticle-based drug delivery strategy is promising for clinical translation in the treatment of GN sepsis.

[0105] Synthesis and characterization of physical properties of TD nanoparticles and nano-PMB: A structure-based approach using peptide chemistry is performed to develop a series of TD (including PEG5kCAsC174, PEG5kOAsC174, PEG5kPAsC174, PEG5kSAsC174) with different anionic charges and hydrophobic moieties. Each telodendrimer (TD) contains a long polyethylene glycol (PEG) chain with eight short branches of negative charge moieties (e.g., carboxylic acid) and four branches of hydrophobic moieties (e.g., heptadecanoic acid or C17). This structure can be easily manipulated by modifying the number of charged branches or altering the hydrophobic moieties. The addition of a branched layer is controlled by the following steps. Initially, all of the active amine sites in the polymer was conjugated by two distinctive protection groups, namely Fmoc and Boc, in a predefined ratio. Then deprotection of the specific Fmoc groups to expose free amine groups which then blocked by hydrophobic heptadecanoic acid. Lastly, the Boc group is specifically deprotected to expose free amine groups, on which different negatively charged groups was conjugated. A ninhydrin test is performed at each conjugation step to confirm the completion of the reactions. The resultant polymers (e.g. PEG5kNegsC174) is then pass through the filtration and dialysis process and are subjected to lyophilization. Finally, the lyophilized blank TD samples is stored at room temperature and characterized by DLS, NMR, and MALDI-TOF MS for determining their physical properties in terms of particle size, molecular structure, and molecular weight, respectively.

[0106] Upon successful construction of the TD NPs, a thin-film hydration technique was applied to encapsulate the antibiotic (PMB) with the desired nanoparticles in a ratio of 1 :10 (w / w), respectively. The weighted PMB then mixed with the NPs individually followed by being placed in a rotating evaporator and strong mechanical pump. Finally, they were reconstitute in the phosphate buffer solution (pH 7.4) and sodium acetate buffer solution (pH 5.5), respectively. To evaluate the shelfstability in the bloodstream, the synthesized nano-PMB samples was stored at room temperature and 4°C in PBS solution (pH 7.4) for four weeks in a row. Then they were characterized by DLS (Fig. 1 B, 1 C), and MALDI-TOF MS (data not shown). Theparticle size of the nano-PMB was almost similar to blank TD. Although the zeta potential value is slightly increased after encapsulation of polycationic PMB to TD. Drug encapsulation was further confirmed by NMR spectroscopy analysis.

[0107] Optimization of the nano-PMB based on PMB loading and release profile: Initially, four nano-PMB were synthesized and were headed for optimization based on their drug-loading stability and sustained release profile. To investigate the PMB loading efficiency of each TD, the nano-PMB equivalent to 1 mg / mL was suspended in 20% fetal bovine serum (FBS), and bovine serum albumin (BSA) separately followed by incubated for 24 hrs at 37°C. Then the samples were placed in agarose gel electrophoresis to analyze their binding affinity to PMB. Preliminary data indicated that nano-PMB is much more stable than the free PMB in the presence of FBS and BSA. The drug release profile of the nano-PMB was also compared with free PMB. The respective samples stored in both physiological pH (pH 7.4) and slightly acidic pH (pH 5.5) were placed in a dialysis membrane and incubated at a 37°C water bath shakerfor 72 hrs. Theirfluoresceine amine intensity will be calculated at each predefined time point to measure the remaining PMB concentrations in the samples. Results showed a slower-release profile of the nano-PMB in both pH conditions than the free PMB; which indirectly indicates the prolonged distribution of PMB in the bloodstream (Fig. 1 D). From data, we observed that after 72 hrs of incubation most of the nano-PMB still show less than 70% drug release whereas free PMB release more than 80% drug after only 6 hrs of incubation.

[0108] Antibacterial efficacy of optimized nano-PMB: The in vitro biological evaluation such as minimum inhibitory concentrations (MIC90) against GN bacteria will be compared with free PMB. The fresh culture of gram-negative bacteria E. coli k12 strain and P. aeruginosa) were co-incubated with different concentrations of nano- PMB, free PMB, and empty NPs in a 96-well plate at a 37°C rotary shaker incubator. After overnight treatment, the growth curve of E. coli K12 and P. aeruginosa were determined using a plate reader.

[0109] Cell cytotoxicity of optimized nano-PMB: We considered that the direct interactions of nano-PMB to cell membrane may have a less toxic effect than the free PMB. To evaluate this, immune cells like PMA-differentiated THP-1 cell lines (human macrophages), RAW 264.7 cells (mouse macrophages), and 293T cell lines (a highly transfectable derivative of human embryonic kidney 293 cells containing SV40 T- antigen) were incubated with the drug separately at 37°C. Then the cell viability of therespective samples were measured by MTT assay. Since the synthetic nano-PMB will ultimately be administered intravenously into the bloodstream, it is reasonable to evaluate the potential hemolytic effect. Human red blood cells (RBC) was used to conduct a hemolysis assay. Here, RBCs were co-incubated for 24hrs on a rotary shaker water bath at 37°C with different concentrations of nano-PMB, and free PMB (Triton X-100 and PBS as control). Then the readings at different wavelengths (420nm, 540nm, and 577nm) were measured using the Nanodrop at predefined time points.

[0110] Investigation of the anti-inflammatory efficacy of optimized nano-PMB and empty nanoparticles: PMB not only act as a potent bactericidal, but they are also well-known for their endotoxin-binding affinity. And LPS inactivation is a major way to reduce overwhelming inflammatory cytokine production. The nano-PMB may have similar LPS-neutralizing potency. We tested this conclusion through in vitro anti-LPS assay of the nanoformulation. We co-cultured the LPS, LTA, PAMP (GN or GP) and DAMP-stimulated RAW 264.7 cells (mouse), PMA-differentiated THP-1 cells (human) and Bone-marrow derived macrophages (primary macrophages) with our formulated nano-PMB, free PMB, and NPs only. After overnight incubation at 37°C, we performed an ELISA to quantify representative pro-inflammatory cytokines such as TNF-a, IL-6 and IL-1 b.

[0111] SEAP reporter TLR Specific cell lines: We quantified TLR4 and TLR2 activation representing LPS bioactivity with HEK-Blue™ hTLR4 and HEK Dual hTLR2 reporter cells, respectively (InvivoGen, San Diego, CA, USA) engineered to produce secreted alkaline phosphatase (SEAP) in response to TLR4 stimulation. The cells were cultured following the manufacturer’s protocol in Dulbecco’s modifed Eagle’s medium without phenol red supplemented with penicillin-streptomycin, L-glutamine, and 10% (v / v) fetal bovine serum. We seeded 50,000 cells per well on a 96-well cell culture plate in 180 pl of cell culture media. We then added 20 pl of samples containing drugs with and without LPS in duplicates to each well for a final working volume of 200 pl. TLR4-signaling inhibitor LPS:RS 5 pg / ml (InvivoGen) was applied as positive control for TLR4 mediated SEAP secretion. Here, PBS served as a negative control. After incubation in 37° C for 24 h, we collected cell culture media from the wells by aspiration. For the QUANTI-Blue detection for SEAP, 20 pl of media sample per well was pipetted on 96-well plates and subsequently 180 pl of QUANTI-BlueTM Solution (InvivoGen), prepared following the manufacturer’s instructions, was added to thewells. Optical densities (OD) at 620 nm were read with a microplate reader (FLUOstar Omega, BMG Labtech, Ortenberg, Germany) after incubating the plates for upto 6 h in 37°C. To correct for possible bias caused by slight variations in the temperature of the plates during incubation, we included a negative human AB serum control for each sample on the plates and subtracted the control OD from the sample readings. TLR4 and TLR2 activation due to LPS was then determined by comparing the data analysis.

[0112] Examining luciferase activity in NF-KB luciferase transgenic mice: Toward our goal to confirm LPS attenuation of the nano-PMB, we used transgenic mice that express luciferase under the control of NF-KB, enabling real-time in vivo imaging (IVIS) of NF-KB activity in intact animals. The NF-KB reporter hemizygous HLL (HIV-long terminal repeat / luciferase) mice (8-12 weeks old) were used for in vivo bioluminescence studies. Their genotypes were confirmed by the presence of NF- B- induced bioluminescence by injection of luciferin (200 yzL / 20 g mice, 150 mg kg— 1 ) when the mice will be 3-4 weeks old at the steady state. One day before the procedure, the abdominal area of the mice were shaved for optimal imaging acquisition. To obtain a basal bioluminescence level of each experimental mouse, luciferin solution were i.p. injected, and 10 min later the abdominal bioluminescence were acquired using IVIS while the mice were under anesthesia through isoflurane inhalation. At the study day, LPS (E. coli, dose of 0.2 mg / kg), LPS+PMB (0.2 mg / kg+4 mg / kg), LPS+nano-PMB (0.2 mg / kg+4 mg / kg), and LPS+NP (0.2 mg / kg+40mg / kg) were i.v. injected into the mice (n=3). PBS saline was injected as a control. The abdominal bioluminescence signals were acquired using IVIS 50 (PerkinElmer) after 2,4, 6, and 24 h post-injection. Blood samples was also collected after 3h to investigate the cytine release.

[0113] With respect to Figure 2.1 , at first, four nano-PMBs were synthesized (through the Thin-film hydration method) and were headed for optimization based on their drug-loading stability and sustained release profile. To investigate the PMB loading efficiency of each TD, the nano-PMB equivalent to 1 mg / mL was suspended in 20% fetal bovine serum (FBS), and bovine serum albumin (BSA) separately followed by incubation for 24 hrs at 37°C. Then the samples were placed in agarose gel electrophoresis to analyze their binding affinity to PMB. Preliminary data indicated that nano-PMB is much more stable than the free PMB in the presence of FBS and BSA. The drug release profile of the nano-PMB was also compared with free PMB.The respective samples were stored in physiological pH (pH 7.4), placed in a dialysis membrane, and incubated at a 37°C water bath shaker for 72 hrs. Their fluoresceine amine intensity will be calculated at each predefined time point to measure the remaining PMB concentrations in the samples. Results showed a slower-release profile of the nano-PMB in both pH conditions than the free PMB; which indirectly indicates the prolonged distribution of PMB in the bloodstream (Figure 2-1 ). From data, we observed that after 72 hrs of incubation, most of the nano-PMB still show less than 70% drug release whereas free PMB releases more than 80% drug after only 6 hrs of incubation.

[0114] In Figure 2.2 we offer that the direct interactions of nano-PMB to cell membranes may have a less toxic effect than the free PMB. To evaluate this, immune cells like PMA-differentiated THP-1 cell lines (human macrophages), RAW 264.7 cells (mouse macrophages), and 293T cell lines (a highly transfectable derivative of human embryonic kidney 293 cells containing SV40 T-antigen) were incubated with the drug separately at 37°C. Then the cell viability of the respective samples was measured by MTT assay. Since the synthetic nano-PMB will ultimately be administered intravenously into the bloodstream, it is reasonable to evaluate the potential hemolytic effect. Human red blood cells (RBC) were used to conduct a hemolysis assay. Here, RBCs were co-incubated for 24 hours on a rotary shaker water bath at 37°C with different concentrations of nano-PMB, and free PMB (Triton X-100 and PBS as control). Then the readings at different wavelengths (420nm, 540nm, and 577nm) were measured using the Nanodrop at predefined time points. The in vitro biological evaluation such as minimum inhibitory concentrations (MIC90) against GN bacteria was compared with free PMB. The fresh culture of gram-negative bacteria (E. coli k12 strain and P. aeruginosa) was co-incubated with different concentrations of nano- PMB, free PMB, and empty NPs in a 96-well plate at a 37°C rotary shaker incubator. After overnight treatment, the growth curves of E. coli K12 and P. aeruginosa were determined using a plate reader. Both of these studies showed less cell cytotoxicity and comparable antibacterial efficacy of the nano-PMB. The MIC90 values (2 to 8 ug / mL for both bacteria) of each nano-PMB are comparable with free PMB indicating that nano-PMB possesses similar bactericidal potency as free PMB (Figure 2-2) . That means still nano-PMB can kill the GN bacteria in the same mechanism as free PMB. Due to the interaction of PMB with phospholipids, it has a damaging effect on the cell membrane and is known to induce dose-limiting cytotoxicity. If the nano-PMB has anyacute toxicity, it is expected to reflect via cell culture and hemolysis assays. From the results, less cytotoxicity is detected at a concentration of up to 500 ug / mL for the free NPs. All the nanoformulations except one (OA-PMB) showed a greater EC50 value than free PMB for either cell line (Figure 2-2). As only less than 8 ug / mL concentration of nano-PMB is enough for the effective treatment of GN bacteria during in vitro studies, it’ll supposed to reduce the dose-limiting cytotoxicity of free PMB in a future animal study and will increase the probability of safe administration at a required dose. PMB concentration that results in 50% cell viability after 72 hrs exposure is defined as effective concentration (EC50). In the case of human macrophages and kidney cell lines, all of the nano-PMB have better EC50 than free PMB (Figure 2-2). In contrast, Raw cells showed one exception for TD(OA):PMB (Figure 2-2). Less than 20% hemolytic percentage is considered to be acceptable and no potential hemolysis is observed in our preliminary studies with the nanoformulations as well as free PMB (Figure 2.2).

[0115] With respect to Figure 2.3, optimized nano-PMB to cell membrane may have a less toxic effect than the free PMB. To evaluate this, immune cells like PMA- differentiated THP-1 cell lines (human macrophages) and RAW 264.7 cells (mouse macrophages) were incubated with the drug separately at 37°C. Then the cell viability of the respective samples was measured by both MTT assay and LDH release assay. Both of these studies indicate the safe dose of nano-PMB compared to the free PMB.

[0116] Due to the interaction of PMB with phospholipids, it has a damaging effect on the cell membrane and is known to induce dose-limiting cytotoxicity. If the nano-PMB has any acute toxicity, it is expected to reflect via cell viability and LDH release assays. From the results, less cytotoxicity is detected at a concentration of 50 ug / mL for the nano-PMB compared to free PMB.

[0117] In Figure 2.4 released endotoxins from the killed GN bacteria can stimulate cytokine storm which induces a secondary immune response during sepsis. To evaluate the endotoxin-neutralizing efficacy of the nanoformulation, in vitro anti- LPS assay were performed in Raw 264.7 cells. We observed notable efficiency of cytokine reduction in mouse macrophages by all of the nano-PMB as well as free PMB. Since one of the empty NP denoted as ‘TD(CA)’ showed significant anti-inflammatory activities. As a result, we have selected this TD NP for further anti-inflammatory evaluation. Later, PMA-differentiated THP-1 cells (human macrophage) were challenged by LPS (50 ng / mL) and co-cultured with PMB (4 ug / mL), TD(CA):PMB(equivalent to 4 ug / mL PMB) and TD(CA) (40 ug / mL), separately. Here, non-treated supernatant with phosphate-buffered saline was used as a negative control, and only LPS-treated culture was used as a positive control. After overnight incubation at 37°C, the cell culture medium were harvested to separate the supernatant and an enzyme- linked immunosorbent assay (ELISA) was performed to quantify representative pro- inflammatory cytokines (TNF-a and IL-6) levels. Our data confirmed that both the drug and empty NP can inhibit cytokines levels against the representative GN bacterial LPS with great efficiency (Figure 2.4).

[0118] Since bacterial protein and peptides are indicated as ligands only for membrane TLRs, specifically for our studies, we used genetically modified HEK-Blue hTLR4 and hTLR2 cells that overexpressed the selected receptor. In these cells, the binding of a specific ligand to the receptor results in NF-KB activation and expression of the reporter enzyme secreted embryonic alkalic phosphatase (SEAP). HEK-Blue cells were stimulated overnight by the appropriate specific ligands (LPS, or LTA for TLR4, and TLR2, respectively) with and without free PMB, TD(CA):PMB, TD(CA) NP only and LPS:RS (a commercially available TLR4-inhibitor). After this, the SEAP activity was colorimetrically analyzed in the post-culture media and indicated activation of the particular TLR by a specific ligand. As shown in Fig. 2-3, the high absorbance level was noticed only for HEK-Blue hTLR4 cells stimulated with LPS. All other groups with either PMB or our nano-PMB showed similar reduction of absorbance. Activation of HEK-Dual hTLR2 was observed in response to LTA (Fig. 2-3). Here, PMB and LPS:RS were not able to reduce the activation of hTLTR2 cells against LTA, although our TD(CA):PMB and TD(CA) Np only can still reduce the NF-kb activity significantly (Fig. 2-3). These observations clearly indicate that our nano-PMB and free nanoparticle can inactivate both TLR4 and TLR2 receptors and control NF-KB and AP-1 pathway activity.

[0119] In Figure 2.5 we evaluated the endotoxin-neutralizing efficacy of the nanoformulation, in vitro anti-PAMP assays were performed in THP-1 cells against different TLR and non-TLR ligands from both gram-negative (E. coli and P. aeruginosa) and gram-positive (S. aureus) bacteria. We observed notable efficiency of cytokine reduction in human macrophages by the nano-PMB and empty TD NP in comparison to PMB only. Since PMB can only neutralize LPS (GN bacterial PAMPs), it didn’t show any cytokine inhibition against LTA (GP bacterial PAMPs), whereas both nano-PMB and TD NP can significantly reduce cytokine stimulation. Here, non-treatedsupernatant with phosphate-buffered saline was used as a negative control, and only LPS-treated culture was used as a positive control (Figure 2.5).

[0120] Since non-TLR ligands can also induce hyperinflammation, we used TNFa (as a non-TLR ligand) in THP-1 cells and genetically modified HEK-Blue hTLR4 cells that expressed TNFa receptors. In these cells, the binding of a specific ligand to the receptor results in NF-KB activation and release of TNFa (THP-1 cells) or expression of the reporter enzyme SEAP (hTLR4 cells). These observations clearly indicate that our nano-PMB and free nanoparticle can inhibit TNFa interaction with TNFa receptors in both THP-1 and TLR4 cells and control NF-KB and AP-1 pathway activation.

[0121] In addition, the DAMPS molecule (another non-TLR ligand) can also be inactivated from cytokine stimulation of the nano-PMB and empty TD NP (Figure 2.5).

[0122] In Figure 2.6 we confirmed LPS attenuation of the nano-PMB, we used transgenic mice expressing luciferase under the control of NF-KB, enabling real-time in vivo imaging (IVIS) of NF-KB activation in intact animals. The NF-KB reporter hemizygous HLL (HIV-long terminal repeat / luciferase) mice (8-12 weeks old) were used for in vivo bioluminescence studies. Their genotypes were confirmed by NF-KB- induced bioluminescence by luciferin injection (200 / zL / 20 g mice, 150 mg kg- 1 ) when the mice were 3-4 weeks old at the steady state. One day before the procedure, the abdominal area of the mice was shaved for optimal imaging acquisition. To obtain a basal bioluminescence level of each experimental mouse, luciferin solution was i.p. injected, and 10 min later the abdominal bioluminescence was acquired using IVIS while the mice were under anesthesia through isoflurane inhalation. At the study day, LPS (E. coli, dose of 0.2 mg / kg), LPS+PMB (0.2 mg / kg+4 mg / kg), LPS+TD(SUA):PMB (0.2 mg / kg+4 mg / kg), and LPS+NP (0.2 mg / kg+40mg / kg) were i.v. injected into the mice (n=3). PBS saline was injected as a control. The abdominal bioluminescence signals were acquired using IVIS 50 (PerkinElmer) after 2,4, 6, and 24 h post-injection. Blood samples were also collected after 3h to investigate the cytokine release.

[0123] After analysis we observed attenuated LPS-induced NF-KB activation as indicated by the reduced bioluminescent signals in the abdominal area from LPS- challenged mice treated with NP only in comparison to the positive control (Figure 2.6). We also showed a significant reduction of cytokines by the nano-PMB and TD NP in compared to free PMB.

[0124] In Figure 2.7 released LPS can stimulate cytokine storm which induces a secondary immune response during sepsis. To evaluate the endotoxin-neutralizing efficacy of the nanoformulation, in vitro anti-LPS assay were performed in healthy human whole blood followed by sepsis patient whole blood. We observed significant cytokine reduction in both healthy human and patient whole blood by the nano-PMB as well as free PMB. Interestingly the empty NP denoted as ‘TD(SUA)’ showed similar significant anti-inflammatory activities. Our data confirmed that both the drug and empty NP can inhibit cytokines levels against the representative GN bacterial LPS with great efficiency (Figure 2.7).

[0125] With respect to Figure 2.8, we evaluated signaling transduction pathways associated with TNF-a and IL-6 expression in macrophages by immunoblotting studies. Upon the recognition of LPS to TLR4 via MD-2 co-receptors, dimerization of TLR4 causes the production of a complex LPS-TLR4-MD2 and activates the downstream signaling via MyD88 dependent or independent manner. In the MyD88-dependent pathway, the NF-KB pathway is essential for regulating inflammatory gene expression. When the NF-KB family, including p65, is in the cytoplasm and binds with IKB, it activates the transcription and expression of cytokines and adhesion molecules. These are closely related to inflammation and the immune response. When stimulated by pro-inflammatory factors like LPS, I KB kinase (IKK) phosphorylates I KB, leading to NF-KB nuclear translocation and further activation of inflammatory genes. Our studies showed that LPS activates NF-KB and its signaling pathways, while TD(SUA):PMB and TD(SUA) restrain this activation. This suggests that the NF-KB may be part of the mechanism by which the TD NP exerts its effects. Six hours after LPS treatment, the phosphorylation of NF-KB / p65 and IKBa increased compared to controls. TD significantly suppressed the phosphorylation of this molecule in Raw 264.7 cells (Figure 2.8). Western blot results showed that LPS treatment induced the phosphorylation of p65 and IKBa (Figure 2.8). Since our TD can competitively inhibit LPS-TLR4 interaction, it can block the dimerization of TLR4 and consequently both TD(SUA):PMB and TD NP can significantly suppress the phosphorylation of p65 and IKBa and then inhibit the production of pro-inflammatory cytokines (TNF-a and IL-6).

[0126] In Figure 2.9, we evaluated signaling transduction pathways associated with TNF-a and IL-6 expression in macrophages by immunoblotting studies. Upon the recognition of LTA to TLR2 / 6 receptors, hetero-dimerization of TLR2 activates thedownstream signaling via MyD88 dependent pathway, where, the NF-KB pathway is essential for regulating inflammatory gene expression. When the NF-KB family, including p65, is in the cytoplasm and binds with IKB, it activates the transcription and expression of cytokines and adhesion molecules. These are closely related to inflammation and the immune response. When stimulated by pro-inflammatory factors like LPS, IKB kinase (IKK) phosphorylates I KB, leading to NF-KB nuclear translocation and further activation of inflammatory genes. Our studies show that LPS activates NF-KB and its signaling pathways, while TD(SUA):PMB and TD(SLIA) restrain this activation. This suggests that the NF-KB may be part of the mechanism by which the TD NP exerts its effects. Six hours after LPS treatment, the phosphorylation of NF- KB / p65 and IKBa increased compared to controls. TD significantly suppressed the phosphorylation of this molecule in Raw 264.7 cells (Figure 2.9). Western blot results showed that LPS treatment induced the phosphorylation of p65 and IKBa (Fig. 2-9C- E). Since our TD can competitively inhibit LTA-TLR2 / 6 interaction, it can block the dimerization of TLR2 / 6 and consequently both TD(SUA):PMB and TD(SUA) NP can significantly suppress the phosphorylation of p65 and IKBa.

[0127] In Figure 2.10, we evaluated signaling transduction pathways associated with TNF-a and IL-6 expression in macrophages by immunoblotting studies. Upon the recognition of TNF-a to TNF-a receptor, it activates the downstream signaling, where, the NF-KB pathway is essential for regulating inflammatory gene expression. When the NF-KB family, including p65, is in the cytoplasm and binds with IKB, it activates the transcription and expression of cytokines and adhesion molecules. These are closely related to inflammation and the immune response. When stimulated by pro- inflammatory factors like LPS, IKB kinase (IKK) phosphorylates IKB, leading to NF-KB nuclear translocation and further activation of inflammatory genes. Our studies show that LPS activates NF-KB and its signaling pathways, while TD(SUA):PMB and TD(SUA) restrain this activation. This suggests that the NF-KB may be part of the mechanism by which the TD NP exerts its effects. Six hours after LPS treatment, the phosphorylation of NF-KB / p65 and IKBa increased compared to controls. TD significantly suppressed the phosphorylation of this molecule in Raw 264.7 cells (Figure 2.10). Western blot results showed that LPS treatment induced the phosphorylation of p65 and IKBa (Figure 2.10). Since our TD can competitively inhibit TNFa-TNFaR interaction, and inhibit downstream signaling, as a result, bothTD(SUA):PMB and TD(SUA) NP can significantly suppress the phosphorylation of p65 and IKBa.

[0128] Table 1-1 presents the charaterization of TDs including particle size, zeta potential and CMC.

[0129] Figure 2.11 prsents Imipenem, which is a type of antibiotic belonging to the carbapenem group, which works by inhibiting the synthesis of bacterial cell walls. However, it's unable to neutralize the endotoxins released by the bacteria it kills, which can lead to an inflammatory response. To address this, we tested our TD NP pretreatment to inhibit the hyperinflammation caused by imipenem treatment. First, we incubated E. coli DH5a with imipenem (I Ml) for 24 hours at 37°C and collected theresulting cell lysates (also known as PAMPs). We then measured the protein concentration of these lysates and added 50 ug / mL of the PAMPs to fresh Raw 264.7 cells, with and without pretreatment of our TD NP (at 250 ug / mL or 500ug / mL), and incubated them at 37°C. After 4hr and 24hr, we collected the supernatant for cytokine analysis (ELISA). Our results showed a significant reduction in cytokines in TD NP- treated groups compared to the non-TD NP-treated group, indicating that our TD NP could be beneficial in controlling hyperinflammation caused by other traditional antibiotics, like IMI.

[0130] With respect to Figurer 2.12, we present an MTD study, in which C57BL / 6J mice (8-12 weeks) were injected intravenously (i.v.) with PMB 5mg / kg (selected MTD for PMB from the reference) (n=3), 7.5mg / kg (n=3) and 10mg / kg TD(PMB) 5mg / kg (n=1); TD(SUA):PMB 5mg / kg (n=3), 7.5mg / kg (n=3) and 10mg / kg (n=1 ); TD(SIIA) only 250mg / kg (n=3) and 500mg / kg (n=3) for both single dose and multiple-dose up to seven days (single dose each day). We monitored the survival of the mice following i.v. injection. We found mice with a single dose of PMB 7.5mg / kg and 10mg / kg were dead immediately after injection, whereas all of the mice survive after both single and multi-dose of TD(SUA):PMB 7.5mg / kg and 10mg / kg. Although both PMB and TD(SUA):PMB with 5mg / kg can survive up to seven days of multi-dose treatments. So, the maximum tolerated dose for free PMB and TD(SUA):PMB was measured at 5mg / kg and 10mg / kg, respectively. We also analyzed the safety assessment of the nanoparticles through blood chemistry results including blood urea nitrogen (BUN) level (an indicator of kidney toxicity) and alanine transaminase (ALT) level (an indicator of hepatotoxicity) where we observed PMB (5mg / kg) treated mice surpassed the normal range of both parameters whereas TD(SUA):PMB (5mg / kg and 7.5mg / kg) treated mice showed normal level of BUN and ALT.

[0131] Table 1-2. IC50 (pM) of TDs for 50 ng / mL LPS induced TNF-a production in RAW 264.7 and THP-1 cells, 100 ng / mL LPS activated HEK-TLR-4 and THP-1 Blue cells, and 1 pg / mL LTA activated HEK-TLR-2 cells.

[0132] Table 1-4. IC50 of TDs for 10 ng / mL LPS induced TNF-a production in healthy whole human blood for 2 h.

[0133] Table 1-5. IC50 of TDs for 100 ng / mL LPS induced TNF-a production in mouse bone marrow derived macrophage (BMDM) cells for overnight.

[0134] With respect to Figure 2.13, we present: pathologically, that the heart and kidney show no injury in all groups, but acute tubular epithelial injury or ATN can be histologically normal but show increased serum creatinine. Though the PMB mouse showed elevated BUN levels whereas the nano-particle treated showed normal BUN, it can also be convincing data that nano-particles can be kidney-protective. However, from a histology standpoint, there were no kidney injuries observed in all groups. There are differences in liver among all groups. Free PMB-treated mice showed focal liver necrosis with neutrophils (Fig. 2-14), in the setting of no infection model, which can be due to drug injury to the liver. Neutrophils also show focally in the TD(SUA):PMB 7.5 mg / kg group but no necrosis. There was no inflammation or necrosis in other groups. So, elevated ALT levels were also observed in the groups with histological evidence of injury.

[0135] In Figure 2.16, the infected animals were treated with TD and TD nanoformulations of PMB and imipenem (IMI) antibiotics and free drugs. Blood was collected at different time point for cytokine analysis. It indicates that I I / IC treatment induce more cytokine (TNF-a and IL-5) production in vivo than PBS treatment and PMB group, which can be inhibited by the co-administration of TD nanodrug. In addition, TD nanodrug alone can also inhibit cytokine production similarly as other nano-antibiotic treatment groups Statistical significance was done by multiple comparison tests following one-way ANOVA (****p<0.0001 ; ns, no significant difference). TD in Figure 2.16 refers to TD(SUA) with a detailed structure of PEG5kSUA8C174 : (SUA refers to succinic acid).

[0136] The results presented herein demonstrate the in vitro safety and efficiency of the Nano-PMB for direct killing of GN bacteria and inactivating broadspectrum septic molecules (e.g., PAMPs and DAMPS) in both mice and human cell lines. Thus, the nanoformulations successfully evaluated herein for in vivo toxicity and subjected to the CLP model in mice, which involves polymicrobial infection and thus represents a clinically relevant septic state are promising for clinical treatment of GN sepsis by effective control of both infection and hyperinflammation.

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Claims

CLAIMS:Claim 1 . An immune modulating nanodrug comprising the formula: P-B(X)-(R®m-L-Rn)x, wherein P is a hydrophilic oligomer or polymer; B is a branching structure and (X) is a branching unit; R® is at least one negatively charged moiety and all R® are the same; L is an optional linker group or a bond; R is at least one hydrophobic moiety; m is integer from 1-16; n is integer from 1-16; and x is integer from 1-8.Claim 2. The immune modulating nanodrug of claim 1 , wherein P is a polyethylene glycol (PEG) polymer having an average molecular weight from 44 Da to 40 kDa.Claim 3. The immune modulating nanodrug of claim 1 , wherein P is a dendritic zwitterionic polymer.Claim 4. The immune modulating nanodrug of claim 1 , wherein P is a dendritic zwitterionic glycerol phosphorylcholine polymer.Claim 5. The immune modulating nanodrug of claim 1 , wherein the B is a nonlinear branching structure having one or more branching monomer unit X.Claim 6. The immune modulating nanodrug of claim 5, wherein the B and X are each at least one amino acid having more than one amine group.Claim 7. The immune modulating nanodrug of claim 6, wherein the at least one amino acid is selected from the group consisting of lysine, ornithine, 2,3- bisaminopropanoic acid, 3-(Bis(3-aminopropyl)amino)propanoic acid, 4,5- bisaminopentanoic acid, 2,4-bisaminobutanoic acid, 3,4-bisaminobutanoic acid, and 3,5-Bis(aminomethyl)-benzoic acid).Claim 8. The immune modulating nanodrug of claim 1 , wherein R® is at least one negatively charged acid.Claim 9. The immune modulating nanodrug of claim 8, wherein R® is at least one selected from the group consisting of carboxylic acid, succinic acid, itaconic acid (ITA), squaric acid, oxalic acid, sulfuric acid, and phosphoryl acid (PA).Claim 10. The immune modulating nanodrug of claim 1 , wherein the L is at least one selected from the group consisting of a polyethylene glycol moiety, an aminoacid, a polyamino acid, a polyserine moiety, an enzyme cleavable peptide moiety, a disulfide bond moiety and an acid labile moiety, a polyglycine moiety, a poly(serine- glycine) moiety, an aliphatic amino acid moiety, a 6-amino hexanoic acid moiety, a 5- amino pentanoic acid moiety, a 4-amino butanoic acid moiety, and a beta-alanine moiety.Claim 11. The immune modulating nanodrug of claim 1 , wherein R is a saturated fatty acid or an unsaturated fatty acid.Claim 12. The immune modulating nanodrug of claim 1 , wherein R is at least one selected from the group consisting of ethyl (C2) , propyl (C3), butyl (C4), pentyl (C5), hexyl (C6), heptyl (C7) t2-propypentanoyl (PPA), 4-phenylbutyl (PBA), nonanoyl (C9), heptadecanoyl (C17), lauroyl (C12), myristoyl (C14), palmitoyl (C16), oleoyl (C18), cholesteryl (CHO), vitamin E succinyl (VE), riboflavin glutaryl (Rf), cholic acetyl (CA), and fluvastatin (Flu).Claim 13. The immune modulating nanodrug of claim 1 , wherein said nanodrug comprises immune modulating activity by altering immune cell response in infection and inflammation.Claim 14. A method of modulating immune responses in a mammal by inhibiting the activation of a Toll-like receptor (TLRs), said method comprising administering a therapeutically effective amount of the nanodrug of claim 1 , to a mammal in need thereof.Claim 15. The method of claim 14, wherein said nanodrug inhibits said Toll-like receptor signaling stimulated by damage associated molecular patterns (DAMPs) and pathogen associated molecular patterns (PAMPs).Claim 16. The method of claim 15, wherein the Toll-like receptor (TLR) is a TLR-4 receptor and / or TLR-2 receptor.Claim 17. The method of claim 15, wherein DAMP / PAMP for TLR stimulation are endotoxins.Claim 18. The immune modulating nanodrug of claim 1 , wherein said nanodrug is selected from the group consisting of: PEG5kSUA8C174, PEG5kSA8C174, PEG5kOA8C174, PEG5kPA8C174, PEG5kSUA8C124, PEG5kSUA8C144, PEG5kSUA8C164, PEG5kSUA8C184, PEG5kSUA8CHO4, PEG5kSUA8VE4, PEG5kSUA8Rf4, PEG5kSUA8FIU4, PEG20kSUA8C174, PEG2kSUA8C174,GPCsSUAsC174, PEG5kSUA8-L-C174, PEG5kSUA4C174, PEG5kSA4C174, PEG5kOA4C174, PEG5kPA4C174, PEG5kITA4, PEG5kITA8, PEG5kITA8C174, and PEG5kITA8-L-C174.Claim 19. A method of modulating an immune response in a mammal by stimulating a Toll-like receptor, said method comprising administering a therapeutically effective amount of the nanodrug of claim 1 , to a mammal in need thereof.Claim 20. The method of claim 19, wherein the nanodrug is at least one selected from the group consisting of PEG5kSUA8C44, PEG5kSUA8PPA4, PEG5kSUA8PBA4, PEG5kSUA6C176, and PEG2kSUA6C176Claim 21 . A method of modulating an immune response in a mammal by inhibiting the activity of cytokine for cytokine receptor activation, said method comprises administering a therapeutically effective amount of the nanodrug of claim 1 , to a mammal in need thereof.Claim 22. A method of modulating an immune response in a mammal by inhibiting cytokine activity via lipid raft interruption, said method comprises administering a therapeutically effective amount of the nanodrug of claim 1 , to a mammal in need thereof.Claim 23. A method of modulating an immune response in a mammal, said method comprising, administering a therapeutically effective amount of the nanodrug of claim 1 and stimulating immune cells that inhibit tumor necrosis factor-alpha (TNF- a) and lnterleukin-1 (IL-1 b) activity in a mammal in need thereof.Claim 24. The method of claim 19, wherein the nanodrug is at least one selected from the group consisting of: PEG5kSUA8C174, PEG5kSA8C174, PEG5kOA8C174, PEG5kPA8C174, PEG5kSUA8C184, PEG5kSUA8CHO4, PEG5kSUA8VE4, PEG20kSUA8C174, PEG2kSUA8C174, GPC8SUA8C174, PEG5kSUA8-L-C174, PEG5kSUA4C174, PEG5kSA4C174, PEG5kOA4C174, and PEG5kPA4C174.Claim 25. A nanocarrier composition comprising multiple self-assembled units with the formula:P-B(X)-(R°m-L-Rn)x,wherein P is a hydrophilic oligomer or polymer; B is a branching structure and (X) is a branching unit; R® is at least one negatively charged moiety and all R® are the same; L is an optional linker group or a bond; R is at least one hydrophobic moiety; m is integer from 1-16; n is integer from 1-16; and x is integer from 1-8.Claim 26. The nanocarrier composition of claim 25, wherein P is a polyethylene glycol (PEG) polymer having an average molecular weight from 44 Da to 40 kDa.Claim 27. The nanocarrier composition of claim 25, wherein P is a dendritic zwitterionic polymer.Claim 28. The nanocarrier composition of claim 25, wherein P is a dendritic zwitterionic glycerol phosphorylcholine polymer.Claim 29. The nanocarrier composition of claim 25, wherein the B is a nonlinear branching structure having one or more branching monomer unit X.Claim 30. The nanocarrier composition of claim 29, wherein the B and X are each at least one amino acid having more than one amine group.Claim 31 . The nanocarrier composition of claim 30, wherein the at least one amino acid is selected from the group consisting of lysine, ornithine, 2,3- bisaminopropanoic acid, 3-(Bis(3-aminopropyl)amino)propanoic acid, 4,5- bisaminopentanoic acid, 2,4-bisaminobutanoic acid, 3,4-bisaminobutanoic acid, and 3,5-Bis(aminomethyl)-benzoic acid).Claim 32. The nanocarrier composition of claim 25, wherein R® is at least one negatively charged acid.Claim 33. The nanocarrier composition of claim 32, wherein R® is at least one selected from the group consisting of carboxylic acid, succinic acid, itaconic acid (ITA), squaric acid, oxalic acid, sulfuric acid, and phosphoryl acid (PA).Claim 34. The nanocarrier composition 25, wherein the L is at least one selected from the group consisting of a polyethylene glycol moiety, an amino acid, a polyamino acid, a polyserine moiety, an enzyme cleavable peptide moiety, a disulfide bond moiety and an acid labile moiety, a polyglycine moiety, a poly(serine-glycine) moiety, an aliphatic amino acid moiety, a 6-amino hexanoic acid moiety, a 5-amino pentanoic acid moiety, a 4-amino butanoic acid moiety, and a beta-alanine moiety.Claim 35. The nanocarrier composition of claim 25, wherein said nanocarrier composition comprises at least one therapeutic compound.Claim 36. The nanocarrier composition of claim 25, wherein the assembled nanocarrier composition encapsulates at least one positively charged therapeutic compound selected from the group consisting of: an antibiotic peptide, a lysozyme, a cytocrhom c, a growth factor, a fibroblast growth factor (FGF), a vascular endothelium growth factor (VEGF), an interlukin-4 (IL-4), an interlukin-10 (IL-10), a interferon-r (INF-r), a tumor growth factor-p (TGF-P), a chemokine, and an interlukin- 8 (IL-8).Claim 37. The nanocarrier composition of claim 25, wherein the nanocarrier composition encapsulates at least one therapeutic compound selected from the group consisting of: polymyxin B, polymyxin A, aminoglycosides, gentamycin, Daptomycin, and doxorubicine,Claim 38. The nanocarrier composition of claim 25, wherein the nanocarrier composition encapsulates at least one therapeutic hydrophobic compound selected from the group consisting of: an anticancer drug, an antibiotic, an anti-inflammatory drug, paclitaxel, docetaxel, curcumin, imipenem, doxycycline, ceftaroline, methyl itaconate (ITA), and erythomycin.Claim 39. The nanocarrier composition of claim 25, wherein the nanocarrier composition chelates at least one platinum drug compound selected from the group consisting of: cisplatin, carboplatin, and oxaliplatin.Claim 40. A method of modulating an immune response for the treatment of bacterial infection, inflammation, sepsis and cancer, in a mammal in need thereof, said method comprising administering a therapeutically effective amount of the nanocarrier composition of claim 25.

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