Esterase responsive release of anti-cancer agents from conjugated lipid nanocarrier and the regulatory considerations

ABSTRACT

The release of active agents in tumors rather than normal tissues, limits systemic exposure and toxicities. Targeting over-expressed esterase enzyme in the tumor microenvironment can selectively release immune-active agents like Programmed Death-1 (PD-1) and PD-1 ligand inhibitors from ester-sensitive lipid nanocarriers, offering a novel approach compared with conventional therapies. PD-1 and PD-L1 association cause T-cell inactivation, whereas blocking their association improves their cytotoxic mechanism. The patent application US2022/0080051-A1 discloses a novel immune-active agent conjugated with lipid to form a nanocarrier for esterase-sensitive release. These nanocarriers selectively enter leaky vasculature of tumors through enhanced permeability and retention effect, undergo ester cleavage to release agents, and are reported to increase bioavailability by 24 times. Further, with other agents or alone it achieves targeted synergistic cancer therapy. Also, the current patent spotlight delves into the crucial formulation considerations necessary for obtaining successful approval of lipidic nano products from relevant regulatory authorities.

Plain language summary

Executive summary.

• A novel lipidic nanocarrier platform with prodrug approach was created to deliver immune-modulating prodrugs to tumor sites specifically.

Discussion

• The over-expressed esterase enzyme in the TME triggers the release of active agents from the lipidic nanocarrier within tumors, minimizing exposure to normal tissues and reducing systemic toxicities.

Conclusion

• The combination of synthesized PD-1 or PD-L1 inhibitor prodrug along with TLR agonists and A2aR antagonists demonstrated a notable impact on local tumor shrinkage and cytotoxic tumor killing.

Future perspective

• Further research is needed to explore its potential for precise tumor-targeted release and broader therapeutic applications in cancer as well as some other disease management.

1. Background

Cancer is the next most common cause of death worldwide, trailing only cardiovascular disorders. According to the “Cancer Facts and Figures 2023” released by the American Cancer Society, the second most common cause of cancer-related deaths in women is breast cancer. Only lung cancer claims the lives of more women annually. About 297,790 new cases of invasive breast cancer will be diagnosed in women, and about 43,700 women will die from breast cancer [1]. Female breast (2.26 million cases, 11.7%), lung (2.21 million, 11.4%), and prostate cancers (1.41 million, 7.3%) accounted for the majority of these total cases [2]. PD-L1 is expressed in 20% of triple-negative breast cancers (TNBCs) [3]. PD-L1 is a biomarker of the prognosis of prostate cancer and also it is expressed in 20% of TNBCs [4]. Blocking PD-L1 with an anti-PD-1 antibody in various cancers, like melanoma and lung cancer, showed positive responses in 6–17% of patients. The existing cancer treatment options include surgery, radiation, and chemotherapy, sometimes all together. Chemotherapy, which targets fast-growing cells like cancer, can also harm healthy cells, causing strong side effects. Therefore, developing alternative cancer treatments with fewer side effects is a priority due to current medication drawbacks. Using prodrugs, which are inert molecules converted into active drugs after administration, increases chemotherapy selectivity and reduces systemic toxicity and overall drug side effects [5]. The specific delivery to the targeted tissue is one of the challenges in traditional drug delivery systems. Non-selective treatments may affect healthy tissues and organs, leading to systemic toxicity. Strategies such as targeted therapies, immunotherapies, and nanocarrier systems aim to enhance selectivity, minimize off-target effects and improve the overall safety and efficacy of cancer treatments. In oncology, surgery is the most common therapeutic approach. But even with improvements in equipment and surgical technique, up to 30–40% of those patients experience recurrences of cancer within 5 years, primarily as a result of localized tumor cells that remain in the surgical margins [6]. The novel treatment of cancer includes a rational combination of immunotherapies that offer mechanisms to both activate and maintain immune responses in the face of tumor-derived immune checkpoint mediators. The introduction of checkpoint inhibitors, CAR-T-cell treatments, and other immunotherapies has resulted in an improvement in cancer patient survival rates [7]. The prodrugs have grabbed major attention because of their high specificity and reduced side effects. Thus, nanocarriers offer a promising solution by delivering immune-modulating prodrugs selectively to the tumor site, thereby addressing the challenge of achieving effective immunotherapy.

Although in recent eras there have been great advancements in cancer therapy and improvement in survival rates, still there is a requirement to explore novel treatment approaches encompassing a broad range of therapeutic pathways due to the diverse nature of cancer. Such a need is particularly required in dealing with solid tumors located in anatomically important areas, for example, glioblastoma, squamous carcinoma of the head and neck, and lung adenocarcinoma. In these cases, treatment options may be restricted to standard radiotherapy and/or chemotherapy [8]. Although neoadjuvant chemotherapy can be given before surgery or intense radiation therapy. The benefits of chemotherapy before surgery include the capacity to reduce tumor volume, help in the control of micrometastatic illness, and enhance operability. Locally and regionally advanced solid tumors, that may benefit from neoadjuvant therapy, are frequently treated with definitive surgical resections, either with or without radiation and systemic therapy [3]. Even though surgery remains a primary treatment for most solid tumors, still local regional recurrence after surgery is the primary cause of treatment failure in solid tumors. Hence, there is a strong need for targeted treatment of tumors. Tumor microenvironment (TME) has become an important factor in tumor immunotherapy. Generally, the esterase enzyme is present in high concentrations in the TME. Thus, targeting it can offer a potent solution. The current patent application describes explicitly the enzymatic cleavage of lipidic nanocarrier with an ester group-containing immune-active agents in the presence of over-expressed esterase for selective delivery at the tumor site. The above instance emphasizes the importance of developing novel approaches to combat these difficult cancers. A surface receptor called PD-1 which is over T-cell serves as its checkpoint. With the initiation of such a checkpoint, it leads to T-cell collapse. There are several types of PD inhibitors, including monoclonal antibodies, mRNA [9] and other small molecules, peptides and peptidomimetic compounds, macrocyclic peptides [10]. PD-1 and its ligands PD-L1 & PD-L2 are two of the most extensively studied immune checkpoints in cancer. This protein receptor is a member of the CD28 superfamily and is encoded by the B7-H1 and B7-DC. PD receptor-to-ligand interaction resulted in, inhibitory signals sent to neighboring cells. When PD-1 binds to its ligand PD-L1 additional signaling pathways, that prevent T cells from becoming activated are triggered. PD-L1 is majorly present on the surface of tumor cells. The binding of PD-L1 on the tumor cell surface to PD-1 on the T-cell surface provides inhibitory signals that induce T-cell apoptosis while suppressing T-cell activation and proliferation, allowing tumor cells to avoid immune elimination [11]. Due to its long-lasting response, low toxicity, and remarkable clinical efficacy, the PD1/PD-L1 pathway inhibition has attracted the attention of numerous researchers in recent years to work on the targeted delivery for the effective management of tumors [12].

Generally, tumors have hyper-permeable vasculature and poor lymphatic drainage, which passively increases macro-molecule retention in the tumor. This is referred to as the enhanced permeation and retention (EPR) effect. The EPR effect is caused by two factors, in other words, increased permeability of blood vessels in tumors and limited lymphatic drainage from tumor tissues. This leads to the accumulation of large molecules in tumors. Depending upon the EPR effect, anticancer nanoparticles can accumulate largely in tumors [13]. Nanotechnology is often considered superior to conventional formulations in drug delivery and drug targeting due to its high drug entrapment efficiency, solubility, permeability, and penetration capacity [14]. Currently, cancer nanotechnology has emerged as a viable approach to deliver anticancer medications, offering potential advancements in cancer treatment. Nanocarriers are highly capable of transporting anticancer drugs in the TME due to the EPR effect. Nanocarriers are useful in delivering drugs to other inaccessible sites of the body and particularly to specific targets due to their small size. Site specificity has a significant therapeutic advantage because it enhances the drug's delivery at the site of action to the maximum if not all [15]. Nanoparticles exhibit a size from 1 to 1000 nm, and they confer enhancements to both the bioavailability and selectivity of anticancer drugs [16]. Lipid-based nanoparticles (LBNPs), including solid lipid nanoparticles (SLNPs), liposomes, and nanostructured lipid carriers (NLCs), have garnered significant interest in the areas of drug discovery and cancer treatment [17]. These nanoparticles possess the capability to effectively transport both hydrophobic and hydrophilic molecules. Nanomaterials also provide a platform for developing effective immunomodulators for integrating multiple treatments for combination therapy [18].

Moreover, they exhibit minimal to no toxicity while extending the drug's duration of action through extended half-life and controlled release mechanisms. The incorporation of lipophilic drugs into the lipid bilayer and also drugs of opposite polarity in the aqueous interior can be made possible by using nanocarriers having both polarities [19].

Numerous stimulus-responsive nanoparticles were developed to achieve stability transitions by utilizing differences in TME features such as elevated reactive oxygen species (ROS) lower pH, altered Glutathione concentrations, and over-expression of enzymes like esterase [20]. A new type of drug delivery system that releases drugs specifically in cancer cells, contains a nano assembly comprised of the polymeric prodrug in conjugation with novel trivalent phenyl-boronate moieties for drug conjugation via ether linkages, as well as β-lapachone. While ether linkage enables no drug release under physiological conditions, the β-lapachone molecules can induce the ROS burst particularly in cancer cells due to the presence of (NAD(P)H: quinone oxidoreductase-1 specifically. This enzymatic catalysis triggers cleavage of ether bonds via ROS generation, resulting in efficient drug release in cancer cells. This nano assembly shows strong anticancer effects and can be adapted for other drug delivery systems [21]. Recently, Ya Liu et al. also reported it by using nanocarrier vehicles, they have maximized the anti-tumor effect by combining an immune active agent with a chemotherapeutic agent [22]. The dual-delivery strategy enhanced the innate and adaptive immunity synergistically, greatly increasing animal survival.

Nanocarriers have the propensity to deliver antigens, and immune-modulatory agents such as Toll-like receptor (TLR) agonists, and Adenosine A2a Receptor (A2aR) antagonists to immune cells for combined chemo-immunotherapy. These therapeutic strategies can activate both adaptive and innate immune responses to induce cancer cell death. Ming Shi et al. stated that TLRs in tumor cells do have dual biological effects on tumor cell growth and their existence. TLR5 is highly expressed in breast cancer and gastric carcinoma cells. TLR5 signaling suppresses breast cancer growth while promoting gastric cancer cell proliferation. TLR5-deficient breast tumor cells promote tumor growth, whereas TLR5 agonist flagellin hinders tumor growth by improving apoptosis and reducing the proliferation of tumor cells [23]. In a study by Leone et al., the combination treatment of anti-PD-1 and CPI-444, a novel inhibitor of CTLA4 resulted in a significant improvement in tumor regression and overall animal survival in both the CT26 and MC38 tumor models. The result was especially noticeable in the CT26 tumor model, where combination therapy improved the rate of complete tumor elimination from 20% in mice treated with anti-PD-1 alone to 70% in mice treated with CPI-444 and anti-PD-1 together. These findings confirmed that combining CPI-444 with anti-PD-1 therapy can result in a remarkable enhancement in the body's immune response against tumors, surpassing the effects of either agent used alone [24]. The approach of using TLR agonists and A2aR antagonists with a conjugated lipidic nanocarrier comprising PD-1 inhibitor prodrug undergoing esterase-responsive cleavage in TME for a significant anti-tumor effect can be an ideal one. In the consideration of above discussion, we found the current patent important and amenable to be highlighted as it describes such an enzyme-triggered delivery of anti-cancer agents. In line with the previous findings, US2022/0080051A1, the current patent application, reports the synthesis of PD-1 inhibitor with carboxylic acid, and alcohol functionality followed by conjugating with cholesterol or phospholipid for a prodrug structure cleavable by over-expressed esterase enzyme in the TME.

2. Patent discussion

The US 2022/0080051 A1 patent application discloses the synthesis of PD-1 inhibitor prodrug with carboxylic acid, alcohol functionality followed by conjugating with cholesterol or phospholipid gives a prodrug structure cleavable by over-expressed esterase enzyme in the TME. The significance of this synthesis extends beyond its immediate application to PD-1 inhibitor development, influencing the broader landscape of cancer therapy. By achieving a more efficient and reproducible synthesis of PD-1 inhibitors, the accessibility and scalability of these crucial immunotherapeutic agents can be enhanced. This holds particular importance in the context of cancer therapy, where PD-1 inhibitors have shown remarkable efficacy in unleashing the immune system to target and eliminate cancer cells. The improved synthesis not only streamlines the manufacturing process but also aids in nanocarrier development via ester linkage and EPR effect. By using novel PD-1 inhibitors in conjunction with modern nanocarrier (prodrugs approach), the overall purpose of more effective treatment, decreased side effects, and enhanced therapeutic utility in the treatment of cancers can be achieved.

A phospholipid is a type of lipid that is found in almost all cell membranes. Phospholipids form lipid bilayers due to their amphiphilic property, which is important for the facile formation of lipidic nanocarriers. The structure of a phospholipid molecule is made up of two hydrophobic fatty acid “tails” and a hydrophilic ‘head’ comprising of a phosphate group that can be modifiable by simple organic compounds like serine, choline, or ethanolamine. Drug-lipid conjugates with biodegradable linkages, like esters, thioesters, and other linkers shown in this invention, make up prodrugs. The utilization of such reversible drug lipid conjugates in the formulation of nanocarriers can pave an attractive pathway in cancer therapy.

Anticancer drugs are often non-selective. Targeting esterase enzyme which is highly expressed in the tumor microenvironment aids in the selective release of active therapeutic agents from prodrugs with ester group via cleavage into tumor tissues to enhance delivery efficiency while minimizing toxicity to normal tissues. This esterase-responsive nanocarrier design offers a revolutionary approach to current cancer treatment methods by providing a more targeted and controlled drug release to the tumor site with fewer side effects. In a study, a hydrophobic tetraphenyl-ethylene derivative was linked to hydrophilic polyethylene glycol using a phenolic ester to get an esterase-responsive amphiphilic polymer. When introduced to an aqueous solution, this polymer self-assembled into nanoparticles with a low critical micelle concentration of 0.53 μM and emitted a strong yellow fluorescence. This polymer efficiently entrapped the chemotherapeutic drug doxorubicin, resulting in a polymer-doxorubicin complex with as high as 21% drug-loading capacity. The esterase enzyme caused polymer-doxorubicin nanoparticles to release doxorubicin in the esterase over-expressed TME [25]. Some of the reactions where the linkage units can be obtained are provided below:

In one example, ‘R’ is a PD-1 inhibitor prodrug that is attached to carboxylic acid functionality (Figure 1) to form a drug-lipid conjugate cleavable by an esterase.

Figure 1.

PD-1 Inhibitor prodrug synthesis scheme with carboxylic acid functionality.

Here, PD-1 inhibitor prodrug synthesized with alcohol functionality (Figure 2)

Figure 2.

PD-1 Inhibitor prodrug synthesis scheme with alcohol functionality.

The current invention describes a drug conjugated with a lipid to obtain a prodrug with an ester linkage unit. The lipid bilayer is composed of cholesterol and the PD-1 inhibitor. The prodrug can be converted to its active form in the over-expressed esterase TME (Figure 3). Further, the nano-encapsulated PD-1 inhibitor prodrug is co-formulated with different immuno-modulatory agents, such as TLR agonists and A2aR antagonists for the effective treatment of cancer.

Figure 3.

Schematic representation of drug release from a conjugated lipid nanocarrier via cleavage, targeting the eradication of cancer cells in the presence of over-expressed esterase.

EPR: Enhanced permeation and retention; TME: Tumour micro environment.

The enzyme cleavable linker such as esterase helps to release the drug specifically in the TME. Esterase-responsive nanoparticles could be sensitive to tumor cells due to the over-expressed esterase enzyme in tumor cells. Esterase-responsive nanocarriers are specially designed to target and accumulate in tumor cells. For clinical relevance, one approach namely adjunctive therapy can be a great hope. In adjunctive therapy, patients can be treated with specialized nanocarriers containing PD-1 Prodrug, combined with chemotherapeutic or pharmaceutical agents. These nanocarriers can be used to target and treat primary cancer effectively. The therapy aims to reduce tumor size, improve survival rates, stabilize the disease, and enhance the patient's health. Additionally, it allows for reduced doses of standard chemotherapy and biological agents, minimizing their side effects and enabling extended treatment. Carbonic esters are also used to connect drugs and carriers to form prodrugs, which are drugs that release quickly when exposed to intracellular esterase in cancer cells.

The current patent application discloses major advantages of lipid-based nano-formulations as bio-compatibility / biodegradability and no general toxicity of the lipid-based formulations; and flexibility and manipulation of size and surface charge based on the required purpose. Also, further, a comparative advantage of esterase-responsive drug release over non-esterase-responsive drug release is provided in Table 1.

Table 1.

Comparative advantages of esterase-responsive drug release over non-esterase-responsive drug release.

Advantages	Esterase-responsive	Non-esterase-responsive
Targeted drug delivery	Enables precise delivery within the tumor microenvironment	May lack specificity, leading to non-targeted drug distribution
Minimized systemic exposure	Reduces exposure to healthy tissues, improving overall safety	This may result in higher systemic exposure, potentially causing toxicity
Enhanced therapeutic efficacy	Controlled release optimizes drug availability at the target site	Release mechanisms may not be optimized, impacting treatment outcomes
Customizable release profiles	Offers flexibility in tailoring drug release according to needs	Limited ability to customize release profiles for specific therapies
Potential for combination therapies	Can be used in combination with other agents for comprehensive treatment	Limitations in combining with other therapies due to release constraints

For this disclosure, lipid-based nano-formulation might have a diameter of 40–150 nm in diameter and a surface charge ranging from -40 to +40 mV; and (iii) the invention's liposomes can contain one or more lipid prodrugs as the liposome's constituent lipid part.

The lipid-based nanoformulation of the present disclosure consists of PD-1 prodrug co-formulated with one or more additional immune-modulating agents, whereby the immune-modulating agents include, Immunogenic cell death (ICD) inducing chemotherapeutic, TLR agonists, CTLA4 inhibitors such as CD122, CD40, CD96, CD73, CD47, CD27, CD28, OX40, JAK, PI3K delta, PI3K gamma, GITR, CSFIR, TAM, arginase, ICOS, A2AR, BTLA, CTLA, CD137 (also known as 4-1BB) and or prodrugs thereof. In the present patent application, liposomes consist of a PD-1 prodrug co-formulated with an ICD-inducing chemotherapeutic such as doxorubicin. Further, liposome-containing PD-1 prodrug co-formulated with a TLR agonist. TR5 is the suitable TLR agonist selected from the group consisting of TR3, TR4, TR5 and TR6.

Lipid drug conjugates are a type of lipid-based drug delivery system that combines the therapeutic benefits of small molecule drugs with lipids targeting abilities. The drug is linked to a lipid molecule, allowing it to be targeted to specific cells or tissues. The lipid conjugation helps in improving the pharmacokinetics of drug delivery as well as protecting the conjugated drugs from hydrolysis and enzymatic cleavage. The combination of lipid-prodrug and liposome has the advantage of increasing the solubility of the prodrug while also encapsulating numerous drugs. Tumor tissues differ from normal tissues in several ways, including increased glutathione levels, over-expressed enzymes, and an acidic pH, which can be used to design nanocarriers. Nanoparticles can deeply penetrate the tissue via the EPR effect to elicit better therapeutic response.

3. Methodology

3.1. Chemical synthesis of PD prodrug comprising cholesterol

A synthetic scheme of PD prodrug is provided in Figure 4. The synthesis of prodrug involves a series of steps:

Figure 4.

Chemical synthesis of PD prodrug comprising cholesterol.

3.2. Step I: Reduction

The starting material, 3-bromo-2-methylbenzoic acid when reacted with borane tetrtrahydrofuran (THF) as a solvent system at 10–25°C for 14 h underwent reduction in which the carboxylic acid group was reduced to alcohol namely 3-bromo-2- methylphenyl methanol (II).

3.3. Step II: Suzuki's reaction

Then Suzuki's reaction has been carried out on compound II for C-C bond formation between the bromide of compound II and boronic acid derivative such as (2,3-dihydrobenzo[b][1,4]dioxin-6-yl)(l4-oxidaneylidene) borane in presence of Palladium complex namely palladium diphenylphosphonoferrocene dichloride [Pd(dppf)Cl₂] under basic (K₃PO₄) condition in THF for 2 h at room temperature to obtain5-chloro-4-((3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-2-methylbenzyl)oxy)-2-hydroxybenzaldehyde (III)

3.4. Step III: Mitsunobu reaction

The compound III when reacted with a phenolic compound namely 5-chloro-2,4-dihydroxybenzaldehyde in the presence of Diisopropyl azodicarboxylate (DIAD), triphenyl phosphine (PPh₃) and THF at 20–25°C for 11 h undergone mitsunobu reaction in which the primary alcohol of III converted to aryl ether to give 5-chloro-4-((3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-2-methyl benzyl)oxy)-2-hydroxybenzaldehyde (IV).

3.5. Step IV: Williamson ether synthesis

The compound IV upon reacting with 3-cyano benzyl bromide in the presence of cesium carbonate (Cs₂CO₃) and Dimethylformamide as a solvent system at 25°C for 12.5 h yielded 3-((4-chloro-5-(3-(2,3-dihydrobenzo[1,4] dioxin-methyl benzyl) oxy)-2-formyl phenoxy) methyl) benzonitrile (V) in which the phenol group of IV got converted to ether.

3.6. Step V: Reductive amination

Further, compound V when reacted with (2R,4R)-4-((tert-butyldimethylsilyl)oxy)pyrrolidine-2-carboxylic acid in the presence of acetic acid as catalyst formed imine, which upon treating with Sodium cyanoborohydride (NaBH₃CN), methanol and dimethylformamide as solvent systems undergone reductive amination to form (2R,4R)-4-((tert-butyldimethylsilyl)oxy)-1-(5-chloro-2-((3-cyano benzyl)oxy)-4-((3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-2-methylbenzyl)oxy)benzyl)pyrrolidine-2-carboxylic acid (VI).

3.7. Step VI: Steglich esterification

The compound VI upon reacting with cholesterol in the presence of DCC (dicyclohexyl carbodiimide) as a coupling agent, DMAP dimethylaminopyridine as a base, and DCM as a solvent system for 16 h at room temperature underwent a coupling reaction in which the carboxylic acid group of VI is coupled with the hydroxyl group of cholesterol to form an ester compound named (3S,8R,9R,10S,14S,17R)-10-methyl-17-(5-methyl hexyl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl(2R,4R)-4-((tert-butyldimethylsilyl)oxy)-1-(5-chloro-2-((3-cyanobenzyl)oxy)-4-((3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-2-methyl-benzyl)oxy)benzyl)pyrrolidine-2-carboxylate (VII)

3.8. Step VII: Tert-Butyldimethylsilylether (TBDMS) deprotection

Deprotection of silyl ether (TBDMS) was carried out using TBAF as a reagent and THF as a solvent system on compound VII at 20°C for 2 h to get the hydroxyl group in the final prodrug structure.

3.9. Preparation & characterization of SLNP-PD-TR5 Solid-Lipid nanoparticle

Regarding the patent, the inventor has synthesized a protected PD prodrug comprising cholesterol. Cholesterol has good biocompatibility and the ability to make membranes stable, easy to functionalize hydroxyl groups, and ability to form liposomes. SLNP comprising PD prodrug is synthesized by solvent diffusion method. SLNP are prepared by lipid stock solution of DSPC, CHOL, DSPE-PEG-COOH was prepared in ethanol. Pluronic F-127 is used as a stabilizer. Because of ease of forming dispersion, adequate stability, efficient entrapment efficiency, and target-specific release properties, SLNP is suitable for incorporating PD-1 inhibitor prodrug.

Incorporating pharmaceuticals into nanocarriers, such as solid lipid nanoparticles (SLNs), offers a promising approach for targeted drug delivery. However, SLNs face challenges in loading hydrophilic drugs due to partitioning effects. To overcome this limitation, lipid drug conjugate (LDC) nanoparticles with a drug loading capacity of up to 33% have been developed. This involves creating an insoluble drug-lipid conjugate bulk through methods like salt production (with a fatty acid) or covalent linking (e.g. to ester or ether). The fact that the lipid matrix in SLN is composed of physiological lipids, which lowers the risk of both acute and chronic toxicity, is a definite benefit [26].

The lipid stock solution of DSPC(1,2-distearoyl-sn-glycero-3-phosphocholine), CHOL(Cholesterol),DSPE-PEG-COOH(1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)]) and a TLR agonist (denoted TR5) in ethanol (20 mg/ml) was prepared. Separately, a stock solution of PD prodrug in DMSO (20 mg/ml) was prepared. This lipid mixture was heated for a few min at 50°C. Similarly, the aqueous phase containing the stabilizer was heated using a hot plate while being constantly magnetically stirred (at 300–400 rpm). Under constant magnetic stirring, the lipid mixture was slowly mixed with the aqueous phase. After mixing was completed, the entire mixture was sonicated for about 5 min in a water sonicator bath and then returned to the magnetic stirrer plate with constant stirring for about another hour. Finally, the solvent was removed by running a dialysis membrane with a cut-off size of 12 KDa against Deionized water (DI) for at least 8 h followed by washing. Finally, the SLNP-PD-TR5 complex was concentrated using a centrifuge device (cut-off size 10 KDa, at 3000 g). The results showed that the nanoparticles had an average size of approximately 106 nm and a Polydispersity Index (PDI) of approximately 0.132. The PDI value <0.2 indicates a homogeneous distribution [27]. Therefore, 0.132 indicates the uniformity of nanoparticles. The zeta potential value between ±40 to ±60 mV shows good stability and over 60 mV shows excellent stability behavior of particles in particular aqueous media [28]. The results showed a negative zeta potential of SLNP-PD-TR5 was approximately -11.2 mV, which indicates strongly cationic and strongly anionic.

3.10. Preparation & characterization of SLNP-PD-AR5-DOX solid-lipid nanoparticle

The synthesis of SLNP by solvent diffusion method involves the preparation of a lipid stock solution of DSPC, CHOL, DSPE-PEG-COOH in ethanol (20 mg/ml). Separately, a stock solution of an AR5 prodrug and PD prodrug (Target 1) was prepared in DMSO. DSPC, CHOL, PD prodrug, AR5 prodrug, and DSPE-PEG were mixed to obtain a liquid mixture (20 mg/ml). The lipid mixture was then heated to 50°C and subsequently, to this mixture, 4.7 mg of doxorubicin was added and dissolved by vortexing for about 5 min. The aqueous phase containing Pluronic F127 (2% w/v) was heated using a magnetic hot plate stirrer with constant magnetic stirring (300–400 rpm). The above lipid mixture with doxorubicin was slowly added to this aqueous phase (Pluronic F127) under constant stirring for 30 min. Once the mixing was completed, the entire mixture was sonicated using a water bath sonicator for about 10 min. Finally, the solvent was removed using a dialysis membrane of cut-off 12 KDa size against DI water which was used as a bulk medium for dialysis and the dialysis was performed for at least 8 h. During the period of dialysis, bulk DI water was changed at least three times (3x). Finally, the SLNP-AR5-PD-Dox was concentrated according to the need (cut-off size 10 KDa, at 3000 g).

Characterization of the SLNP-PD-AR5-DOX was determined using a Malvern Zetasizer. The results showed that the average size of the nanoparticles was approximately 101.7 nm with a PDI of approximately 0.158. The PDI value <0.2 indicates a homogeneous distribution [27]. Therefore, 0.158 indicates the uniformity of nanoparticles. Nanoparticles with a zeta potential between -10 and +10 mV are approximately neutral [29]. The results showed the negative zeta potential of SLNP-PD-AR5-DOX was approximately -10.5 mV.

The linkage of PD prodrug to the nanoparticle and the loading of doxorubicin in the same nanoparticle has not been reported in the patent under discussion.

3.11. Tumor Inhibition of SLNP-PD-TR5 using EMT6 cells in vivo

Tumor inhibition of SLNP-PD-TR5 is determined by using EMT6 cells in vivo. Murine breast cancer EMT6 cells were subcutaneously inoculated in the right rear flank region of Balb/c mice. TR5 at 1 mg/kg, and SLNP-PD-TR5 (a combination of TR5 at 1 mg/kg and cholesterol at 5 mg/kg) were given to the animals. Tumor volumes were measured three times in two dimensions with a caliper and calculated using the formula: $V=L\times W\times W÷2$, where V is tumor volume, L is tumor length (the longest tumor dimension), and W is tumor width (the longest tumor dimension perpendicular to the longest tumor dimension).

The tumor growth inhibition (TGI) was calculated based on the tumor size data of day 21. The results showed treatment with SLNP-PD-TR5 and SLNP-TR5 produced significant anti-tumor activity when compared with the vehicle. The TGI was found to be 74.72% and 83.13%, (p < 0.05).

4. Conclusion

US 2022/0080051 A1 patent application describes the use of lipid-based nanocarriers in the enhancement of solubility and reduction of the toxicity of a compound. Phospholipids are amphiphilic and can form lipid drug conjugates. This ultimately helps in the attainment of a tumor-selective drug delivery system. The drug conjugated to lipid cleaves by an esterase enzyme expressed in the TME and releases the drug into the TME. This provides targeted drug delivery in case of functional groups cleavable by esterase which is over-expressed at the tumor site.

Local delivery of an agent that inhibits PD-1 or PD-L1 via the conjugation with a lipid to a tumor or peri-tumoral region has great potential to become the cornerstone in immunotherapy. It has been observed that local tumor shrinkage and cytotoxic tumor killing are caused by PD-1 or PD-L1 inhibitors in conjunction with TLR agonists and A2aR antagonists. The innate immune system is strengthened in conjunction with these adaptive immune responses.

5. Regulatory perspective

Since the formulation mentioned in the above patent application involves the lipid as a nanocarrier and PD-1 inhibitor as a drug (prodrug), the regulatory approaches for the sustainability of formulation in the regulatory marketing approval become an associated aspect to dive. For safe use of nanoparticle treatments in humans, thorough toxicology assessments are crucial for clinical application. Regulatory bodies recommend careful evaluation of any changes in drug substance, manufacturing process, or formulation during clinical development to ensure product safety. These changes, such as alterations in synthesis, reagents, impurity profile, or manufacturing method, can impact safety and require comprehensive examination throughout the investigational new drug process. Changes in the source material, sterilization method, route of administration, composition, dosage form, manufacturing process, and container closure system can impact product quality. Stringent procedures ensure comparison testing of previous and changed processes to evaluate equivalency, quality, and safety. If materials are not comparable, additional studies are needed to support safety and bioavailability in proposed trials. To support the safety and bioavailability of the material to be used in the proposed trials, sponsors should carry out further qualification and/or bridging studies when analytical data show that the materials generated before and after are not similar [30].

Concerning guidance entitled “Liposome Drug Products Chemistry, Manufacturing, and Controls; Human Pharmacokinetics and Bioavailability; and Labeling Documentation” introduced in April 2018 by the Center for Drug Evaluation and Research USFDA, the lipids that are naturally sourced (e.g., egg lecithin) should be provided with the lipid composition as a range of percentages for each stated lipid present in the mixture and its fatty acid composition. Since the current patented invention involves the usage of phospholipids it is of utmost importance to consider the guideline entitled “Liposome Drug Products Chemistry, Manufacturing and Controls; Human Pharmacokinetics and Bioavailability; and Labeling Documentation”. This guidance provides information on how applicants should submit NDA and ANDA to get product approval. It also states the difficulty in obtaining adequate batch-to-batch repeatability because of the polydispersity issue. Sterilization of lipidic products is also challenging due to their complicated physical and chemical properties. Furthermore, the regulation of several large-scale process variables, including solvents, temperature, pH, surfactants, sterility, and the ratios of polymers to lipids and drugs, to secure regulatory approvals was discussed. In the case of synthetic and semisynthetic lipid distearoylphosphatidylcholine (DSPC), or dimyristoylphosphatidylcholine (DMPC), proof of structure, including fatty acid composition and positional specificity should be provided.

Specification information for every lipid component utilized in the manufacturing of the product such as an identity test capable of distinguishing the intended lipid component from lipids with the same structures, assay based on impurity testing, stability indicating analytical procedure and other testing (degree of unsaturation of the fatty acid side chains, counter ion content and limits on divalent cations, when appropriate) should be included.

Stability studies should address the microbiological, physical and chemical stability and the integrity of the drug product. The evaluation of stability is essential as it assesses the robustness of lipid-based nanocarriers, as it directly influences the overall quality and performance of the product. Lipids, especially unsaturated fatty acids, are susceptible to oxidative degradation. Additionally, both saturated and unsaturated lipids can undergo hydrolysis. Stress testing is useful to know potential degradation and for the determination of stability indicating method. stability studies play a pivotal role in assessing and enhancing the reliability, safety and longevity of lipid-based nanocarriers.

From a regulatory perspective, it is imperative to conduct stability studies and stress testing [e.g., after exposure to high (e.g., 50°C) and low (e.g., -20°C) temperatures, light, pH, and oxygen] for each lipid used in manufacturing, that were used to determine the degradation profile, to develop appropriate stability indicating analytical procedure and to establish appropriate storage conditions and retest period [31].

Despite the clear potential of these nanocarriers is clear, rigorous validation of in vivo applications is required for successful clinical translation. In general, the regulatory approval based on the showing of the benefit-to-risk ratio aspects. However, demonstrating this benefit-to-risk ratio can be challenging in novel technologies like lipid-based nanocarriers due to various reasons like damage to blood cells, inflammation, stress on cells, issues with cell structures and functions, genetic damage, and irritation to skin and eyes. Chronic toxicities, which involve long-term effects, are even more complex to study. It is advised that cytotoxicity tests be performed as part of the in vivo characterization of nanocarriers. To overcome these challenges, researchers can adopt various strategies. One such strategy is to optimize the composition of the nanocarriers to minimize their toxicity and improve their efficacy by using generally regarded as safe excipients. Another strategy is to standardize the characterization methods to enable comparison of results across different studies. Additionally, researchers can use in vitro and in vivo models to assess the safety and efficacy of the nanocarriers. Finally, researchers can also explore the use of computational models to predict the behavior of the nanocarriers in vivo [32].

Regulatory approval can be sought when appropriate toxicity studies are carried out based on the risks. As per the European Union, if the particle size is reduced to <100 nm, then, it is considered as hazardous and thus should accord the “Registration, Evaluation, Authorization and Restriction of Chemicals” (REACH) guidelines [33]. The “REACH Implementation Project on Nanomaterials” (RIPON) recommended that chemical safety assessment (RIPON3) and information requirement (RIPON2) be the two most important facets of nanomaterials. According to their physicochemical characteristics and related risks, nanoparticles are categorized using RIPON2 [34]. RIPoN. If a substance is carcinogenic, mutagenic, or a reproductive toxin, investigations on its effects on reproduction, repeated dose toxicity, sensitization, and carcinogenicity should be conducted. Studies on the genotoxicity, carcinogenicity, and inflammability of the nanoproduct should be conducted if it is composed of redox-active metals, or is photoreactive, or is contaminated with biologically active pollutants. If the nanoproduct has a highly charged surface that is acidic/basic or can cause inflammation and cytotoxicity, acute toxicity as well as repeated dosage toxicity has to be performed. Assuming the nanoproduct doesn't produce any harmful or reactive ions, it's deemed non-toxic. Therefore, based on the nature additional respective characterizing studies have to be done as per the guideline [33].

The size of drug delivery systems impacts how drugs move in the body. Nanocarriers with a size of ≤150 nm can enter or exit specific capillaries in the cancer microenvironment. Larger nanocarriers (100–150 nm) stay within capillaries in normal arteries, while smaller particles (20–100 nm) may disperse to organs like the bone marrow, spleen, and liver. The particles under 10 nm are filtered by the kidneys and not reabsorbed. Nanocarriers from 50 to 200 nm typically cannot escape continuous blood capillaries due to tissue and capillary pore size limitations [35].

The primary objective of Guidance entitled “Pharmacokinetic-Based Criteria for Supporting Alternative Dosing Regimens of Programmed Cell Death Receptor 1 (PD1) or Programmed Cell Death Ligand 1 (PDL1) Blocking Antibodies for Treatment of Patients with Cancer” introduced in December 2022 by the Oncology Center of Excellence USFDA, is to provide recommendations related to different dosage schedules for cancer patients' PD-1 or PD-L1 blocking antibodies. In particular, it highlights the importance of pharmacokinetic factors in supporting alternative dosing strategies for these immunotherapeutic agents. To establish efficacy in the clinical trial, a reference dosing regimen is used for comparison. During pre-approval, the reference regimen for comparison is the dose regimen utilized in early clinical research to evaluate the product's pharmacokinetics and effectiveness.

It proposes undertaking pharmacokinetic studies to find exposure-response relationships and determine the best appropriate dose schedule. Pharmacokinetic studies should consider factors include patient demographics, disease characteristics, concomitant medications, and laboratory data. The guidance also suggests that pharmacokinetic data can support alternative dosing regimens if they provide similar exposure to the approved dose and do not compromise efficacy or safety. The guidance also recommends the consideration of clinical factors such as disease progression, response, and toxicity when determining the need for alternative dosing schedules. As a result, it is critical to examine regulatory considerations as well as other related ares in order to successfully introduce the product into the market [36].

6. Future perspective

In the future paradigm, the inventor proposes the conjugation of immune active agents with lipids to develop prodrugs capable of self-assembling into a liposomal bilayer. These can be utilized in both chemotherapeutic and immunotherapeutic interventions. The current patent application paves an ideal pathway to deliver novel immune-modulating prodrugs specifically to tumor sites with minimal side effects in healthy organs. By selectively targeting tumor cells with high concentrations of esterase compared with generally used anti-tumor nano pharmaceuticals, reducing drug release in normal cells is the major advantage of esterase-responsive delivery of anti-cancer agents. However, this approach is one of the several stimuli-responsive methods and is not yet suitable for clinical use due to over-expression in other diseased cells. Also, the activity of esterase in tumor cells varies from person to person [37].

The usage of ligands and contrast agents in a single conjugated nanocarrier may make them versatile. For instance, solubilization of cheap and easily scalable ligands like lactisole which is capable of binding to circulating tumor cells along with the contrast agents in the lipid matrix of esterase responsive nanocarrier gives the theranostic approach via the single unit [38]. Further immobilization of tumor recognition molecular elements and entrapment of azobenzene compounds into the lipid bilayer membrane which undergoes photoisomerization followed by light irradiation of specific light & wavelength can release the active moiety at the targeted site [39]. In a study, Karp et al. created an enzyme-responsive hydrogel that responds to inflammation. The amphipathic ascorbyl palmitate with esterase-cleavable ester linkage was used to create hydrogel microfibers that could encapsulate the anti-inflammatory drug dexamethasone. In two in vitro murine colitis models, it was shown that these inflammation-responsive hydrogels could selectively cling to inflammatory epithelial surfaces and exclusively release medication upon enzymatic breakage. Compared with regular dexamethasone (Dex) enemas, using Dex-loaded hydrogel enemas every other day in mice significantly reduced inflammation. These hydrogel enemas also led to lower Dex levels in the bloodstream, reducing overall exposure to the drug. Similarly, a group developed a hydrogel for arthritis treatment by combining a special molecule with corticosteroid triamcinolone acetonide (TA). This hydrogel released the drug when it came into contact with specific enzymes or synovial fluid from patients with rheumatoid arthritis. A single injection of this hydrogel with TA suppressed arthritis activity in the treated area, outperforming the equivalent dose of free TA [40].

Mesoporous nanoparticles, such as porous silicon and porous silica, offer advantages like large pore volume, high surface area, and a tunable structure for drug loading. Their hierarchized mesopores make them ideal for constructing hierarchical vehicles. To address quick and uncontrollable drug release from these nanoparticles, medications can be encapsulated in pH-responsive polymers. Hydrogels containing small molecules with enzyme-labile bonds, like triglycerol monostearate and acorbyl palmitate, enable gradual drug release in response to degradative enzymes like matrix metalloproteinases (MMPs) and esterases, enhancing control over drug delivery to the targeted intestinal segment [41].

A polypeptide vesicle responsive to esterase enzymes was designed for controlled drug release. This innovative drug delivery platform demonstrated a selective impact on cancer cells with high esterase expression while sparing normal fibroblasts due to their low esterase activity. The nanovesicle significantly improved in vivo antitumor efficacy and decreased systemic toxicity compared with free DOX at equivalent doses. These results indicate the potential of this enzyme-responsive polypeptide vesicle as a potential, safe, and effective drug delivery system in cancer chemotherapy [42].

Even though the esterase-based delivery of anti-cancer agents via conjugated lipid nanocarriers seems to be an ideal one, the other pathological conditions with esterase and exact tumor-targeted release have to be explored further to make them next-generation therapeutic agents for effective management of cancer.

Financial disclosure

The authors have no financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Competing interests disclosure

The authors have no competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

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No writing assistance was utilized in the production of this manuscript.