Lipid-Based Nanoparticles as a Pivotal Delivery Approach in Triple Negative Breast Cancer (TNBC) Therapy

Round 1

Reviewer 1 Report

In the present review, the authors have exhaustively compiled the different aspects of lipid-based formulations reported for the betterment of triple-negative breast cancer.  The authors have nicely shaped the article by starting from the cancer statistics followed by the pathophysiology of the disease and then the lipid-based approaches reported in this area. With the help of figures, the authors have nicely shown the different types of LPNs and the different mechanisms by which they are absorbed to enhance the bioavailability of poorly soluble drugs. The authors have nicely compiled different types of nanoparticles along with their recent clinical status. Overall, the manuscript is very informative and can be very interesting for the IJMS audience. Although authors have included most of the conventional lipid-based systems yet the inclusion of exosomes, which are also lipidic vesicles (biological vesicles), will further improve the horizon of the present manuscript and hence authors are advised to include a paragraph on exosomes. Authors are also suggested to check the uniformity of the terms used in the manuscript (for eg. SLNs should be used at every place instead of SLN; NLCs should be used everywhere instead of NLC). Authors are also advised to check uniformity at other places as well (Line 230 and 245 either use the space before and after the ± or don’t use it throughout the manuscript). As mentioned in the title, this work explicitely compiles the data on cancer some of the FDA-approved formulations given in table 4 which are not related to cancer can be removed.

Author Response

Comment: Although authors have included most of the conventional lipid-based systems yet the inclusion of exosomes, which are also lipidic vesicles (biological vesicles), will further improve the horizon of the present manuscript and hence authors are advised to include a paragraph on exosomes. Authors are also suggested to check the uniformity of the terms used in the manuscript (for e.g., SLNs should be used at everyplace instead of SLN; NLCs should be used everywhere instead of NLC). Authors are also advised to check uniformity at other places as well (Line 230 and 245 either use the space before and after the ± or don’t use it throughout the manuscript). As mentioned in the title, this work explicitly compiles the data on cancer some of the FDA-approved formulations given in table 4 which are not related to cancer can be removed.

Response: The suggestions are well taken.

• In the revised manuscript, we have added a segment in “section 2” depicting the role of exosomes as a lipid-based nanoparticles for the treatment of TNBC.

 

2.6 Exosomes

Exosomes are nanosized extracellular vesicles that are enclosed by lipidic bilayers with diameter ranging from 30 – 150 nm, and are released by almost all kinds of cells [39]. In this context, our lab has isolated exosomes from bovine milk with particle size of 75±0.6 nm [91, 92], and colostrum with particle size 59±1.1 nm [93]. The isolated exosomes further exhibited an increased therapeutic efficiency of anticancer agents to the targeted site [94, 95]. They are basically generated by two invaginations of the plasma membrane. Exosomes are comprised of various surface proteins that are specific to the endosomal pathway, and can enclose nucleic acid, receptors, cytosolic proteins, and drugs [96, 97]. The lipidic layer of exosomes varies from other types of extracellular vesicles like apoptotic bodies, and microvesicles as they are enriched in cholesterol and diacylglycerol [45, 98]. Exosomes are considered one of the encouraging natural carriers of antineoplastics or biomolecules as they bypass their elimination through circulation and enhance cell-specificity towards cancer cells after modification via surface proteins [99]. It was observed that exosomes are biodistributed via body fluids to transport drugs or biomolecules to the cancer cells within the vicinity or dwelling remotely, which offers an advantage in recognizing potential pathological situations. The exosomes follow various uptake mechanisms namely direct membrane fusion, or endocytosis [100].

Naseri, et al., 2018 isolated exosomes from bone marrow-derived mesenchymal stem cells, loaded with locked nucleic acid (LNA)-modified anti-miR-142-3p oligonucleotides (MSCs-Exo) to diminish the expression of miR-142-3p in 4T1 breast cancer cell lines. It was observed from the in-vitro and in-vivo results that the MSCs-Exo showed efficient delivery and enhanced penetration of anti-miR-142-3p in to breast cancer cells respectively alongwith increased transcription of regulatory target genes [101]. Gong, et al., 2019 isolated exosomes from human leukemia monocytic cell line (THP-1), and co-loaded with doxorubicin hydrochloride (Dox), and cholesterol-modified miRNA (Cho-miR159) to treat TNBC. Further to increase the targetability of co-loaded exosomes, the system was further conjugated with modified version of a disintegrin and metalloproteinase 15 (Co-A15-Exo) (Figure 7 I). It was observed that flow cytometry data that the A15-Exo exhibited increased cellular uptake (78.60%), as compared to Exo (15.23%). Also, Co-A15-Exo showed enhanced apoptosis in MDA-MB-231 cells as compared to Dox-treated group. Further, Co- A15-Exo showed increased inhibitory rates of tumor volume (92.8%), as compared to Dox (49.5%), and Cho-miR159 (53.7%), revealing a potent synergism among Dox, and Cho-miR159 (Figure 7 II) [102]. Yu, et al., 2019 fabricated erastin-loaded HFL-1 (human fetal lung fibroblast) derived exosomes conjugated with folic acid (FA) to target TNBC cells with overexpressed FA receptors (Erastin@FA‐exo). It was observed that Erastin@FA‐exo increased the cellular uptake of erastin into MDA‐MB‐231 cells, compared to free erastin, Also, Erastin@FA‐exo exhibited significant inhibition of TNBC cells proliferation and migration and promoted ferroptosis alongwith depletion of intracellular glutathione and ROS production [103]. Li, et al., 2020 developed c-Met binding protein conjugated engineered exosomes for the treatment of TNBC. The author has developed Doxorubicin (Dox) loaded polymeric nanoparticles and incorporated them into the macrophages derived exosomes. It was observed that the engineered exosomes exhibited increased cellular uptake of Dox (2.28 times, and 3.31 times), as compared to free-Dox, and Dox-loaded polymeric nanoparticles respectively. Further, the engineered exosomes showed increased apoptosis rate (39.73%), as compared to free Dox (10.58%), and Dox-loaded polymeric nanoparticles (11.33%) [104]. In this context, it is a noteworthy development that our lab has also developed paclitaxel and 5-fluorouracil loaded exosomes, isolated from bovine milk and surface conjugated with folic acid for offering an effective treatment regimen against breast cancer. It was observed that developed exosomes showed an average particle size of 80-100 nm, with 82% entrapment efficiency. Also, the surface functionalized loaded exosomes showed increased cellular uptake, and higher apoptotic index, compared to free drugs [97]. 

• Also, as mentioned by the reviewer, the typographic irregularities within the manuscript have been corrected, and table 4 has been modified in the revised manuscript.

Author Response File: Author Response.docx

Reviewer 2 Report

This is a very interesting review focusing on lipid-based nanoparticles in nanomedicine targeting triple-negative breast cancer (TNBC). Although the nanoparticle-based strategies in Table 1 and Figure 1 are noteworthy, it is regrettable that how those strategies resulted in the proof-of-concept of animal experiments were not clearly described.

1.       Describe the technologies for controlling the size of different types of nanoparticles, loading technologies of water-soluble and lipophilic small drugs, and polymers such as nucleic acids and proteins.

2.       Describe how different types of nanoparticles impact on the drug delivery systems in vivo and in clinical trials. In particular, is it possible to compare and discuss PK, biodistribution, and antitumor effects among different nanoparticles? In particular, the contents related to Figures 1A and B should be described with examples of different types of nanoparticles.

3.       Description about Table 3 is requested in the text.

4.       Table 5 needs the route for administration as well as size of the particles, and the explanation about the route, kinds of drugs and nanoformulation depending on the organs should be added in the text.

 

5.       Please summarize the safety and efficacy of clinical trials.

6.     There are some mistakes or confusion in the text. Please confirm the followings.

1) LNPs are composed of fatty acids ⇒LNPs are composed of lipids

2) Table 2, with reduced drug leaching ⇒with reduced drug leaking

3) In terms of lipid core ⇒In terms of lipid shell

4) p7, amphiphilic ⇒twitterionic

Author Response

Comment: Describe the technologies for controlling the size of different types of nanoparticles, loading technologies of water-soluble and lipophilic small drugs, and polymers such as nucleic acids and proteins.

Response: In the revised manuscript we have added the following paragraph demonstrating various techniques, and technologies employed for controlling the size of LNPs and loading of drugs, nucleic acid, and proteins into the LNPs.

It was observed that the challenging task in the preparation of LNPs is obtaining proper size, and polydispersity of the LNPs and uniform loading of chemotherapeutics within the LNPs. It was observed that the desired shape and size was obtained by controlling the process parameters employed in the various types of preparation procedure of LNPs. For instance, high pressure homogenization is one of the methods employed for the preparation of LNPs. It was observed that during hot high-pressure homogenization, very narrow particle size distribution was obtained, while in cold high-pressure homogenization, a broad size distribution was obtained. It was inferred that the size distribution of LNPs in high-pressure homogenization depends upon the temperature and pressure provided, type of homogenizer employed, and number of homogenization cycles applied. Likewise, in solvent emulsification evaporation method, the particle size of LNPs were controlled by the type and concentration of lipids, and surfactant mixture within the organic phase. It was observed that the particle size ranges between 30-100 nm when the lipid concentration is employed upto 5% w/v, while, above which the particle size increases beyond 100 nm. In case of solvent emulsification diffusion method, the particle size obtained was below 100 nm with narrow particle size distribution. It was observed that particle size increases on usage of non-ionic surfactant, while decreases on using ionic surfactant. However, it was suggested to use combination of two or more surfactants for better control of the particle size. Lastly, in ultrasonication method, the particle size is obtained in the range of 30-200 nm with broad particle size distribution. It was observed that the particle size can be controlled by varying the frequency, intensity and time of ultrasonication [35].  

For the loading of the chemotherapeutics within the LNPs, the active incorporation method can be used i.e., loading of drugs after LNPs formation, or passive i.e., loading of drugs during LNPs formation [36]. Active method involves adsorption or absorption methods that are achieved by incubating the LNPs with concentrated drug solution [37]. Passive method involves mechanical method, solvent dispersion method and detergent removal method [36]. It was observed that the drug loading depends upon the solubility of the drugs within the lipid matrix which is further associated with the composition of the lipid matrix, molecular weight of the drug, the interaction between the drug and lipids and the presence of end functional groups (i.e., ester or carboxyl) in either the drug or lipid matrix [37]. LNPs were also used for the loading of nucleic acid (siRNA, mRNA, and pDNA), proteins. It was observed that fabrication of nucleic acid loaded LNPs include detergent dialysis and ethanol loading technique. However, rapid-mixing method and T-mixing method have gained more popularity as it assures > 90% entrapment efficiency. In recent times, microfluidic mixing approaches were designed based on rapid-mixing approach which further promises to fabricate nucleic acid, and protein loaded LNPs in a more reproducible and scalable fashion. It was further observed that all the mentioned methods allow rapid mixing of lipid containing organic phase into aqueous phase comprised of nucleic acid, and proteins, and resulting in an enhanced entrapment efficiency [38].

Comment: Describe how different types of nanoparticles impact on the drug delivery systems in vivo and in clinical trials.

Response: As suggested by the reviewer, we have added a section in the revised manuscript depicting the fate of LNPs on the drug delivery system in vivo, however, the impact of different types of LNPs in pre-clinical (in-vitro and in-vivo) and clinical trials were already discussed in the “section 3: Clinical status”.

Like cellular uptake, the in vivo fate of the LNPs also plays an important role in the drug delivery system. Basically, the entrapped drug gets released into the physiological surrounding, only after breaking of the lipidic matrix. Such phenomenon occurs via lipolysis wherever lipases are found in abundance, especially in the GIT or surface erosion, in case the lipids are insensitive to lipolysis. It was observed that the LNPs composed of aliphatic esters are rapidly degraded by lipases, especially in the small intestine, while the LNPs comprised of triglycerides were first broken down by lysosomal acid lipases into diglycerides, which was then broken down into monoglycerides, and finally into fatty acids in the GIT, followed by endocytosis. Then, the lipolysates formed and the encapsulated drugs, both are transported to the epithelial surfaces in the form on vesicles or micelles for absorption. It was further observed that apart from GIT, the lipolysis also takes place within tissues, and cells. In the process of erosion, the lipid matrix undergoes either hydrolysis or dissolution which eventually plays a role in complete degradation of lipid matrices based on fatty acids, and are insensitive to lipolysis. It was observed that as the chain length of the fatty acid increases, the rate of erosion of lipid matrix declines, and drug release becomes slow and steady. While, the lipids with medium chain length significantly increases the erosion rate of the lipid matrix, which thereby increases the drug release from the lipid matrix [33]. 

Comment: In particular, is it possible to compare and discuss PK, biodistribution, and antitumor effects among different nanoparticles? In particular, the contents related to Figures 1A and B should be described with examples of different types of nanoparticles.

Response: The suggestion was well taken. As the nanoparticles we discussed in the manuscript are lipid based (LNPs), their PK profile, biodistribution and antitumor effects are almost similar. Hence, no such articles were to be found revealing such comparison. Also, in the figure 1A, and B, we have explained the uptake of LNPs via oral route. So, the uptake phenomenon of the lipids used in different types of LNPs are same. Hence, no further comparisons were found in the uptake mechanism of different types of LNPs.  

 Comment: Description about Table 3 is requested in the text.

Response: As suggested by the reviewer, we have added a description about Table 3 in the revised manuscript.

The various LNPs fabricated in the last decade to enhance therapeutic efficacy against TNBC have been summarized in Table 3.

Comment: Table 5 needs the route for administration as well as size of the particles, and the explanation about the route, kinds of drugs and nanoformulation depending on the organs should be added in the text.

Response: As suggested by the reviewer, we have added the route of administration as well as the particle size of the LNPs in table 5 of the revised manuscript. Also, we have incorporated a paragraph in “section 3” explaining the impact of LNPs on different routes employed for the treatment of different types of cancers.

From table 5, it was observed that most of the LNPs employed in pre-clinical trials for the treatment of different types of cancers were mostly administered via intravenous route followed by oral and inhalation. So, if we just analyse the impact of LNPs over route of administration and targeting tumor site, based on the pre-clinical and clinical trials, we can conclusively state that for targeting lung cancer, pulmonary/inhalational route of administered was considered because the alveolar region of the lungs has larger surface area (~100 m²), extensive vasculature, thin alveolar epithelium (0.1–0.2 μm), and less drug-metabolizing enzymes, which allows enhanced absorption and bioavailability of nanosized LNPs loaded with chemotherapeutics. Also, the mucus membrane present within the alveolar region is composed of phospholipids (lipids) that are the major components of LNPs, as a result LNPs are considered more biocompatible than other types of nanoparticles [155]. Similarly, brain targeting possess a challenge for the delivery of hydrophilic chemotherapeutics, as they were unable to bypass blood-brain-barrier (BBB), hence LNPs were prepared to deliver hydrophilic drugs into the brain. It was observed that LNPs increased the lipophilicity of the drug which facilitates their transportation to brain by crossing the BBB. It was further observed that liposomes can cross BBB via receptor-mediated endocytosis (RMT), which facilitates enhanced accumulation of chemotherapeutics within tumor site. As a result, the off-target side effects were reduced. It was also observed from various studies that for the treatment of brain cancer, LNPs were administered by oral, intravenous as well as intranasal delivery [156]. The cellular uptake of LNPs by the oral route was already mentioned in the “section 2". Briefly, on oral and intravenous administration, the LNPs enters the lymphatic and systemic circulation respectively, after which it targets the brain cancer cells via active (ligand-mediated cellular internalization) or passive (general cellular uptake via EPR effect) targeting [157]. Similar route and the associated approaches were employed for targeting breast, pancreas, and prostate cancer cells [24, 158]. During intranasal administration for brain targeting, the LNPs binds with the mucus layer, which were then taken up by the neurons and translocated in the nerve axons to enter into brain cells, where the LNPs get degraded by the enzymes and drugs get released [156]. LNPs also ensures distinct drug delivery to the lesion site of the colon and rectum for the treatment of colorectal cancer. Most of the LNPs employed for the treatment of colorectal cancer are via oral route. It was observed that the LNPs are absorbed from the intestinal lumen into the circulation of colorectal region through endocytosis or via carrier-mediated transport [159]. Similarly, for treating hepatocellular carcinoma (liver cancer), LNPs are presently under pre-clinical and clinical trials. Like other cancer types, most of the chemotherapeutics are administered orally or intravenously for the treatment of hepatocellular carcinoma. It was observed that after administration of drug loaded LNPs, they non-specifically bind with the serum proteins leading to aggregation and opsonization, and also causes a chance to get blocked within sinusoidal fenestrations. So, to overcome the drawbacks, the LNPs were shielded with PEG, which provide shielding from plasma protein recognition as well as minimized their sizes to < 100 nm in order to cross the sinusoidal fenestrations. Moreover, liver targeting can be achieved by active or passive targeting [160].

Comment: Please summarize the safety and efficacy of clinical trials.

Response: As per suggested by the reviewer, we have added a segment in “section 3” summarizing the safety and efficacy of the clinical trials in the revised manuscript.

The clinical achievement of LNPs with chemotherapeutics, and nucleic acids have revealed the potential of LNPs in the treatment of different types of cancer, However, the number of fruitful products reaching the market does not accurately represent the number of LNPs employed in pre-clinical trials, which indicated that the LNPs still suffers certain challenges while translating from animals to humans. Currently, various strategies have been developed to overcome such challenges. To further improve the stability and protect the drug from leaking, the lipidic structures have been modified that effectively form complex with the encapsulated chemotherapeutic via ionic attraction. Further stability of LNPs in systemic circulation was accomplished by PEGylating the LNPs, which in order safeguard them by reducing their recognition via RES. However, such approach leads to the production of anti-PEG antibodies which reduces the therapeutic efficacy of the LNPs. This incidence results in finding an alternative for PEGylation upon repeated administration. In addition to establish safety and therapeutic efficacy during prolonged circulation, the LNPs must also exhibit enhanced targetability and cellular internalization at the site of action [105]. To accomplish such objectives, the LNPs are fabricated with selective ligands which enables release of drug on targeted site when triggered by aberrations in pH, temperature, oxidation or reduction within the tumor microenvironment [105, 161]

Comment: There are some mistakes or confusion in the text. Please confirm the followings.

1) LNPs are composed of fatty acids ⇒LNPs are composed of lipids

2) Table 2, with reduced drug leaching ⇒with reduced drug leaking

3) In terms of lipid core ⇒In terms of lipid shell

4) p7, amphiphilic ⇒twitter-ionic

Response: As suggested by the reviewer, we have rectified the mistakes or confusion in the revised manuscript.

Author Response File: Author Response.docx

Reviewer 3 Report

This manuscript entitled “Lipid-based Nanoparticles as a pivotal delivery approach in TNBC therapy” deal with the state of arts about different types of LNPs with the latest advances reported for the treatment of TNBC in recent years. Authors are well suited to explain their findings by analyzing many existing literatures. Overall, the manuscript contains originality, so it is recommended that this work can be published in 'Molecular Sciences'.

Author Response

Comment: This manuscript entitled “Lipid-based Nanoparticles as a pivotal delivery approach in TNBC therapy” deal with the state of arts about different types of LNPs with the latest advances reported for the treatment of TNBC in recent years. Authors are well suited to explain their findings by analyzing many existing literatures. Overall, the manuscript contains originality, so it is recommended that this work can be published in 'Molecular Sciences'.

Response: The authors are very thankful to the reviewer for positive recommendations on our manuscript.

Author Response File: Author Response.docx