Liposomal Delivery Systems for Improved Delivery of Docetaxel Against Prostate Cancer

Abstract

Generally, liposomes as a delivery system have been vastly designed for the delivery of therapeutic agents, to overcome various drawbacks, including resistance imposed to various conventional forms of these agents. They are significantly advantageous compared to other kinds of delivery systems because of their ability to deliver hydrophobic and hydrophilic therapeutic agents singly as monotherapy or combined as combinational therapy, owing to bilayer formation. Clinically, liposomes have been applied for delivery of therapeutic agents, with the aim of improving target-site-specificity and reducing toxicity to normal cells or tissues caused by chemotherapeutic drugs, which makes them relatively better options compared to the conventional forms of delivery system. Herein, a critical review which extensively discusses the various liposomal systems of delivery used for docetaxel (DTX) delivery against prostate cancer in the past decade, either alone as mono-therapeutical delivery or in combination with other therapeutic agents is presented. This review affirms to the potentials of employing liposomal systems of delivery for the effective targeted delivery of DTX against prostate cancer.

Introduction

Cancer remains second in position among fatal non-communicable diseases (NCD) in the world, with an alarming number of deaths recorded each year.^1,2 In 2020 alone, it was reported that an estimated 19.3 million new cases of cancer, as well as a high mortality figure of nearly 10 million, were recorded worldwide.^3,4 Cancer continues to impose life-threatening situations to public health generally, and the available coping or treatment strategies against cancer is highly restricted owing to metastasis, which makes the situation a cause of concern.^5,6 There are various types of cancer;inlcuding lung, liver, oesophageal, brain, breast, and prostate, to mention a few.⁷ These various types are known to be of grave concern to the health sector worldwide. Usually, the treatment strategies that are principally employed for cancer treatment include operations, the use of chemotherapeutic agents, hormonal, and radiation therapy. These treatment strategies could be administered as a single treatment or in combination with others like some cases, where chemotherapeutic agents are first used to treat the cancer tumor for the purpose of reducing the mass of the tumor. This is then followed by the use of surgical operation for the removal of the size-reduced tumor which was accomplished by chemotherapy, and lastly, a post-surgery treatment with the use of either chemotherapy or radiation therapy, depending on the requirement.^6,8,9

Prostate cancer is known to be the most frequently occurring type of male cancer.^10,11 Despite the fact that there is improvement in the fight against it as a result of modern technological developments, the dangers and threat posed to the health of men from prostate cancer cannot be underestimated.¹² The rate of survival of prostate cancer patient in recent years has tremendously improved as a result of early detection and therapy, all thanks to the advancement of technology in developed countries.¹³ Generally, upon detection of prostate cancer at the early stage, treatment options such as surgery as well as radiation therapy, have proven to be very effective.¹² It is therefore, not possible to overstate the importance of early detection and treatment of prostate cancer. However, when not detected at an early stage, and the cancer has reachedthe “extra-prostatic or metastatic” stage, an operation or radiation therapy as treatment options has been proven not to be clinically effective against prostate cancer.¹⁰ Owing to the non-effectiveness of surgery and radiation therapy to treat the metastatic prostate cancer, the use of “Androgen Deprivation Therapy” (ADT) or surgical castration became the first point of call for the treatment of metastatic prostate cancer.¹⁴ Regrettably, over time it has become clear that there is no uniform and complete decline in the disease following the use of ADT for treatment, due to the development of castration-resistant prostate cancer by some of the patients.¹² This castration-resistance being developed in some patients could be said to be responsible for the short-term relevance of the use of ADT, which then required the use of other kinds of therapies such as chemotherapy and immunotherapy.^15–18 Recently, there have been significant changes in the therapy of metastatic prostate cancer as a result of several chemotherapeutic drugs being approved such as: docetaxel (DTX), carbazetaxel; new generational hormone therapy: enzalutamide, abiraterone, amongst others.¹² Chemotherapeutic drugs through the use of conventional delivery systems have been used for the treatment of prostate cancer. However, these chemotherapeutic drugs which are delivered using conventional delivery systems, are known to have defective pharmacokinetics; small therapeutic indices; as well as erratic distribution in vivo.¹⁹ Their erratic distribution in vivo results in the target of healthy tissues in the body and this is due to uncontrolled release of the active pharmaceutical ingredients, which in many cases, results in adverse effects such as toxicity.²⁰ Therefore, the importance of finding a good delivery system for these chemotherapeutic drugs with high selectivity and specificity, which can target the metastatic prostate cancer cells and reduce random distribution to healthy cells and tissues to the barest minimum, cannot be overstated.

Nanotechnology has demonstrated possible ways of providing a bridge to the challenges experienced as a result of these conventional delivery systems by providing selective and target-specific delivery systems with small particle sizes within the range of 1 to 100 nm. Nano delivery systems can be designed to have high target specificity; high solubility; stability; and also to reduce toxicity, which plays a crucial role in improving the therapeutic efficacy of therapeutic agents.^21–23 Examples of these nano delivery systems includes hydrogels,²⁴ solid lipid nanoparticles,²⁵ dendrimers,²⁶ micelles,²⁷ liposomes,²⁸ nanocapsules,²⁹ among others. Recently, there has been a wide range of nano delivery systems which have been used to deliver chemotherapeutic drugs in order to maximize their therapeutic efficacy.^1,30–32

Liposomal delivery systems have been developed with the use of nanotechnology. Liposomes are prepared from various phospholipids, structurally consisting of bilayers which surrounds a core (aqueous) that can be used to load hydrophilic drugs. The lipid bilayers are usually used for loading lipophilic drugs, and these lipids are easily compatible and degraded in the biological environment in vivo.^33,34 Liposomes have been applied for the delivery of various active pharmaceutical ingredients against various infections and diseases including cancer.^35,36 As a delivery system for drugs, they are known to possess several advantages such as: (a) the ability to co-deliver both hydrophilic and lipophilic drugs with specificity to their various action sites, as well as synergistically; (b) improve the therapeutic index of the encapsulated drug; (c) shield the encapsulated drug from possible degradation before reaching the target site; (d) improve the half-life and bioavailability of drugs at action sites, and the release of these drugs in a systematically sustained manner; and (e) decrease the toxicity of the drug by causing a change in their mode of distribution and pharmacokinetics within the body.^28,33,37 The application of liposomes as a delivery system to overcome the limitations of the conventional drug delivery systems has been widely studied, with some of the nano delivery systems already approved, while others are in clinical trials stages.^33,38

We are aware that there have been so many review articles on liposomes as a delivery system, prostate cancer, and DTX against cancer. Some of these reviews have focused on the delivery of DTX against lung cancer,³⁹ passive and active targeting of cancer,⁴⁰ immunoliposomes for antibody delivery,⁴¹ and stimuli-responsive liposomes against cancer.¹ Others which focused on prostate cancer, discussed the various liposomal delivery systems explored for the delivery of various chemotherapeutic agents against prostate cancer,²⁰ nanovectorization in the treatment of prostate cancer,¹⁰ target therapy against prostate cancer,¹² and the use of aptamer-drug delivery systems to treat prostate cancer.⁴² Another review focused on the DTX delivery against breast, lung, and prostate cancer.⁶ However, despite the increasing threat to lives by prostate cancer due to resistance to treatment using DTX, which is the approved drug for prostate cancer treatment, and the possible solutions brought about by nano delivery systems, of which liposomes has been one of the most widely explored type of nano delivery systems for DTX, to the best of our knowledge, there has been no review that comprehensively discussed liposomes as a delivery system to improve the therapeutic efficacy of DTX chemotherapeutic agent against prostate cancer within the past 10 years. It was therefore important that we put forward a review of this kind, to update the scientific community on various liposomal delivery systems that have been explored in the last decade, and about the various types of liposomes that have been prepared to improve the efficacy of DTX against prostate cancer, and how they can be improved upon in future.

Herein we proffer some background information in the introduction section about the severity of cancer, prostate cancer, use of conventional delivery systems for the delivery of drugs and their challenges, as well as nanotechnology through the use of liposomes to overcome these challenges. The review goes further, to describe the conventional DTX as a chemotherapeutic agent; it discusses extensively the mechanisms of action of conventional DTX against prostate cancer; the mechanisms of resistance posed to DTX by prostate cancer; as well as the various employed approaches to overcome the resistance posed to DTX by prostate cancer. Subsequently, the review discusses the liposomal delivery system; their classifications; cellular interaction mechanisms; and the methods used in preparing these liposomes. Finally, the review focuses on elucidating the various liposomal delivery systems strategies (mono-therapeutically and in combinational therapy) to improve the anticancer efficacy of DTX against prostate cancer, laying emphasis on the supremacy of liposomes over conventional delivery systems.

Docetaxel; A Chemotherapeutic Agent

DTX is a second generation semi-synthetic anti-neoplastic drug that belongs to the family of the taxoid (Figure 1). DTX is synthesized from a precursor 10 – deacetylbaccarin – 111, which is known to be an inactive compound derived from a plant Taxus baccata.^6,43 DTX is known to be naturally lipophilic, which makes it practically impossible for DTX to be dissolved using water.⁴⁴ However, when compared to its analogue paclitaxel, DTX has a better solubility in water because the chemical structure contains an ester (tertbutyl carbamate) as well as a hydroxyl group which are attached to the phenylpropionate and carbon – 10 respectively.⁶ DTX can induce the physiological inhibition of microtubule depolymerization through its binding and stabilizing activities to tubulin, which causes an arrest of the cell cycle and eventually leads to the death of the cell. It also has the ability to elevate the expression of an inhibitor (p27) of cell cycle and impede anti-apoptotic gene (Bcl-2) expression, conferring on DTX, its anticancer efficacy against a lot of cancer type such as gastric; breast; ovarian; prostate cancer, respectively.⁴⁵ Despite DTX proving to be more effective against cancer cells compared to its analogue paclitaxel, it also has some limitations which in addition to poor solubility in water, includes low selectivity in terms of distribution, as well as a very quick elimination. In addition, the commercially available DTX formulations are known to present severe side effects such as toxicity to the muscular skeleton, neutropenia, stenosis of the nasolacrimal duct, among others.^6,45 These limitations continue to be a source of concern, heralding the urgency for the need of novel delivery strategies to overcome these limitations and thereby maximize the efficacy of DTX against prostate cancer.⁴⁶

Figure 1.

DTX chemical structure.^6,39

Docetaxel Action Mechanism Against Prostate Cancer

DTX anticancer mechanism of action has been established through different pathways which includes the mechanism of inhibiting the process of depolymerization of the microtubules of the cancer cell and the opposition of the effects of the expression of two genes: Bcl-2 and Bcl-xl.⁶ Amongst these two mechanisms of action of DTX against prostate cancer, the microtubules stabilization (depolymerization inhibition) is the most widely accepted, and it entails the binding of DTX to P-tubulin which helps to promote polymerization. This process, causes a disruption of the dynamics of the microtubule, which then affects the functions of the cytoskeletons during the period of cell division.⁴⁷ Given a normal circumstance, the polymerization of these microtubules only occur when guanosine triphosphate and proteins associated with microtubule are present, as it is their sole responsibility to interact with the P-tubulin. However, DTX binds preferentially to this P-tubulin to initiate an artificial assembly (polymerization) of the microtubule, forming a bond which is irreversible, regardless of the presence of the necessary conditions for depolymerization such as calcium ions or low temperatures of 4°C. This impairment of the normal cell cycle progression (known as cell cycle arrest) would lead to anti-proliferation of the cells, which consequently leads a programmed cell death known as apoptosis.^6,47 Also, there have been reports of the family of the taxoids being able to alter functions that are non-mitotic, which includes various signal pathways alterations or intracellular trafficking inhibition. For example, DTX was reported to induce apoptosis through the phosphorylation of Bcl-2, which was accompanied by caspase cascade activation or induce cell anti-proliferation via the downregulation of ERK1/2 signal.^6,47,48 This Bcl-2 protein are known to form a dimer with another protein which are known to be proapoptotic (Bax), and thereby inhibit its function. It has been well established that, prostate cancer cells are usually protected from apoptotic effect that are induced by chemotherapeutic agents in the presence of overly expressed Bcl-2 proteins. Therefore, the phosphorylation of Bcl-2 proteins could help to induce the loss of antiapoptotic effect of these Bcl-2 proteins, which could consequently decrease its binding ability to Bax. Also, the DTX stabilization of microtubule can lead to Bcl-2 phosphorylation, which can cause a direct increase in the amount of free proapoptotic protein (Bax), thus increasing apoptotic activity in the prostate cancer cell.⁶

Docetaxel Resistance Mechanism in Prostate Cancer

Generally, one of the most devastating limitation of chemotherapeutic agents which are used for the treatment of various types of cancer such as prostate cancer, is the development of resistance by these cancer cells to the drug. DTX, just like other chemotherapeutic agents has had its own fair share of this resistance posed by prostate cancer cells, and the various mechanisms have been elucidated and reported. Some of these mechanisms include: (a) the alteration of structures or functions of the microtubule: alterations such as the β111-tubulin upregulation⁴⁹ along with the mutations of either the α or β-tubulin,⁵⁰ or the modification of proteins associated with the microtubule,⁵¹ and could either modify the dynamics of the microtubule or DTX's ability to bind successfully (Figure 2). These mechanisms have been reported to negatively affect the efficacy of DTX^52,53 thereby causing resistance to DTX; (b) the activation of pathways for survival or for escape of the process of apoptosis: This mechanism is accomplished by deregulating various proteins that are associated with signal pathways, which helps to increase the rate of survival of the prostate cancer cells or by allowing the invasion of the process of apoptosis. The activation of pathways such as P13K/AKT^54,55 and MAPK/ERK^48,56 have been reported to be associated with DTX resistance. Apart from these pathways, antiapoptotic proteins Bcl-xl and Mcl-1which belongs to the Bcl-2 family, have been reported to induce resistance to DTX by altering the process of homodimerization of some proapoptotic proteins such as Bax and Bak.^57,58 Also, upregulating the molecules of chaperon such as clusterin^57,59 which plays a major role in the prevention of apoptotic process engendered by chemotherapeutic agents, by isolating or confiscating Bax,⁶⁰ causes resistance in DTX; (c) the upregulation of various drug efflux pumps such as the transporters of adenosine triphosphate-binding cassette (ABC) like6 MRP1 (multidrug resistant protein-1), MRP4 (multidrug resistant protein-4) and MDR1 (multidrug resistant-1), increases the extrusion of DTX, thereby causing resistance in prostate cancer;^11,61–63 (d) by activating antioxidant as an opposing response against the trigger of excessive “reactive oxygen species” (ROS) by chemotherapeutic agents belonging to the taxoid family such as DTX, which usually precedes apoptosis.^47,64 This therefore leads to resistance against DTX in prostate cancer; and (e) by overly expressing the molecules associated with inflammation: Upregulation of the levels of expression of Interleukin-6 (IL-6), “the transforming growth factor”- β1 (TGF- β1), “nuclear factor kappa B” (NF-kB), and the “macrophage inhibiting cytokine-1” (MIC-1) are reported to be associated with DTX resistance.^65–67 .

Figure 2.

Diagrammatic overview of DTX resistance mechanism in prostate cancer. Where AR – “Androgen Receptor”, ARE – “Androgen Response Element,” CAF – “Cancer-Associated Fibroblasts,” DCT – DTX –, EMT – “Epithelial Mesenchymal Transition,” EMT-TF – “Epithelial Mesenchymal Transition Transcription Factors,” TAM – “Tumor-Associated Macrophage,” TME – “Tumor Microenvironment.”⁴⁷.

There have also been the report of blood-prostate-barrier (BPB), which is an inherent physical barrier that causes obstruction between the “prostate stroma” and the “lumen” present in the tube of the prostate gland.⁶⁸ They act dynamically by regulating and controlling to a strict extent, the exchange of substances that occurs between blood and prostate, and is known to limit the penetration of drugs for treatment of prostate related diseases.⁶⁸ This barrier is accomplished by the regulation of the pore sizes of the para-cellular ions, thereby decreasing the ability for these drugs in the blood to penetrate the pore spaces.⁶⁸ The BPB may be comprised of the epithelial cells of the prostatic ductal, the capillaries of the endothelial cells, and the tight junction (Tjs) linking them both.⁶⁹ Claudins are known as the major components of the TJs. These claudins which are expressed widely in prostate cancer (claudin-1; −3; −4; −5; −7; −8; and −10) play crucial roles blood-tumor barrier regulation, which affects the efficacy of chemotherapeutic agents like DTX during chemotherapy.⁷⁰ Therefore, it is of great importance and urgency, that these drugs be formulated using a good delivery system that can navigate through these mechanisms of resistance to DTX, in order to deliver the drug directly into the prostate, and treat the disease of interest such as prostate cancer.

Approaches for Overcoming Docetaxel Resistance in Prostate Cancer

There have been various strategies that have been experimentally investigated for the reduction of resistance posed to conventional DTX against prostate cancer using both the in vitro and the in vivo models. Regrettably, despite so many reported methods being effective in vitro and in vivo, there have either not been sufficient support data clinically or there has been little efficacy shown in one phase or clinical trials or the other.⁶⁷ One of the methods explored to overcome DTX resistance in prostate cancer was the mediation of the expression of pro-inflammatory as well as pro-survival molecules, which are usually found to be up-regulated with increase in resistance.^67,71 For example, NF-kB inhibition with the use of BAY 11–7082 as well as the use of Marchantin M a known anti-inflammatory compound to reduce the expression and inactivation of IL-6 and NF-kB respectively; were reported to decrease DTX resistance in prostate cancer.^72,73 Other methods that have been explored to reduce DTX resistance, has been the target of β-tubulin isoforms and the pathways of ABC-B1 efflux.⁶⁷ From a study which was carried out by Zhu et al, the inhibition of ABC-B1 expression as well as a likely decrease in drug efflux with the use of apigenin (a flavone) was reported to decrease DTX resistance.⁶³ The use of quercetin (a bioactive flavonoid) to suppress the signalling of P13K/AKT was also reported to have reduced the resistance of prostate cancer cells to DTX both in vitro and in vivo.⁵⁵ Another study reported that the co-treatment using bicalutamide and DTX resulted in the significant reduction of the growth of tumor by inhibiting the efflux activity of ABC-B1 in a prostate cancer mouse model.⁷⁴

Just of late, there have been the option of the co-delivery of two or more chemotherapeutic agents, which apparently, can be seen as a very attractive strategy to overcome the resistance posed to DTX, as well as improve its anticancer activity.¹⁰ However, the application of this strategy has been plagued with so many limitations such as the disparities of their independent systemic distributions, pharmacokinetics, along with the rate of clearance of individual chemotherapeutic agents. These limitations have added to the difficulty in actualizing the expected synergism from their maximum individual efficacies, and thus, defeating the aim of co-delivery. As a result, the need for a good delivery system for DTX that could be entrusted with the responsibility of overcoming these limitations, and thus, ensuring maximum pharmacological efficacy for improved anticancer activities against prostate cancer cannot be overstated.

Nanotechnology Approach: Liposomal Delivery Systems

Liposomal delivery systems are structurally closed bilayers that are composed of hydrated phospholipids, which are prepared for the delivery of drugs such as chemotherapeutic agents to cells or tissues of interest. They are known to mimic biological membranes and are generally prepared by using phospholipids with non-homogenous chains which occurs naturally.^33,75 Most of these lipids which are generally used for the preparation of liposomal delivery systems have their origins traceable to humans, and are also pretty much available for sale on a commercial scale. The “Food and Drug Administration” (FDA) have reportedly approved most of these lipids used in the preparation of liposomes such as the “1,2-distearoylglycero-3-phosphocholine” (DSPC), “egg yolk phosphatidylglycerol” (EPG), “hydrogenated phosphatidylcholine” (from soybean), and “1,2-distearoylsn-glycero-3-phosphoethanolamine” (DSPE).³³ Liposomal delivery systems are arguably the simplest to prepare kind of nano systems of delivery that can be utilized in the delivery of a combination of various already in-use chemotherapeutic agents, and thus improving their targeted delivery and specificity.⁷⁶ Generally, there have been a lot of formulations for the treatment of cancer which were made with the use of liposomal delivery systems that have received full approval by the designated authorities in charge, and some of these includes; liposomal doxorubicin for breast cancer in the metastatic stage⁷⁶ and cancer of the ovary,⁷⁷ irinotecan for the treatment of cancer of the pancreas,^78,79 lipoplatin which was approved for breast cancer.⁸⁰

Liposomal Delivery Systems Classification

The classification of liposomes could either be structural based or according to their different classes. The variation in sizes of the various liposomes are dependent on the particular class in which they belong. These sizes in which liposome comes are known to be a determinant factor in their systemic half-life circulation, and thus the size and bilayer numbers have a direct effect in the amount of drug that is entrapped by the liposomes. Structurally, the classification of liposomes can be grouped into three major classes (Figure 3A). On the basis of the lipid layers, liposomes can be classified as follows: (a) “multilamellar large vesicles” [MLVs], which are known to consist of more than five layers and having between 1–15 µm diameter; (b) “oligo lamellar vesicles” [OLVs], which averages bilayers of approximately 2–5 and diameters between 0.1–1 µm; and (c) “unilameller vesicles” [UVs], which consist of various sizes. The class of UVs can further be sub-classified into three different sub-classes and these includes: (i) the small UVs which are known to consist of one lipid bilayer, and possess diameters between 30–70 nm; (ii) the medium UVs whose diameters ranges from 70–100 nm; and (iii) the large UVs whose diameters are above 100 nm.^33,81,82 Liposomal delivery systems can be widely placed into six distinguished categories on the basis of their class, and these includes: (a) the conventional liposomal systems, which are made up of a combination of cholesterol (Chol) and phospholipids that are negatively charged or neutral. Some examples of these liposomes incudes the Daunoxome^® and the Myocet^®;³³ (b) the fusogenic liposomal systems which consist of lipids that can agglutinate with the membrane of the cells like “1,2-dipalmitoyl-sn-glycero-3-phosphocholine” [DOPE];⁸³ (c) the stealth liposomal systems which consist of lipids that have extended bloodstream circulation period;^81,82 (d) the immune-liposomal systems whose surfaces are functionalized using ligands such as antibodies to improve their target-specificity, especially to a receptor that is overly expressed as a result of the disease condition;⁸⁴ (e) the stimuli-responsive liposomal systems such as response to pH, which help to deliver therapeutic agents to the target cell or tissue irrespective of the pH of the immediate environment of that cell or tissue biologically;⁸⁵ (f) the cationic liposomal systems which consist of the combination of lipids that are naturally positive with DOPE, and are most likely to be used for deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) delivery.^33,84

Figure 3.

(A) – Structural classification of liposomal delivery systems; i – MLVs greater than 5 bilayers (1–15µm), ii – OLVs less than 5 bilayers (0.1–1µm), iii – ULVs single bilayer, iv – LUVs (≥100nm), v – MUVs (70–100nm), vi – SUVs (30–70nm). (B) – Cellular interaction mechanisms of liposomal delivery systems: i – liposomal fusion, ii – liposomal adsorption, iii – liposomal lipid exchange, iv – liposomal endocytosis Adapted from Nwabuife et. al, 2021.³³

Cellular Interaction Mechanisms of Liposomal Delivery Systems

Liposomal delivery systems are characteristically known for their ability to release the entrapped therapeutic agent in a systematically controlled manner owing to their well organized bilayers made of lipids. They are also deemed to interact with the cells or tissues of target distinctively through various mechanisms and this include the “adsorption,” “fusion,” “lipid exchange,” or “endocytosis” mechanism as shown below (Figure 3B). The mechanism of liposomal adsorption into cell membrane takes place when there is superiority in the attraction forces such as the “van der Waal forces,” hydrogen bond, and lipophilic insertion as opposed to the repellent forces such as protrusion, steric-hydration and interactions forces that are electrostatic by nature.⁸⁶ The fusion of liposomes tends to occur when there is an interaction between the liposomal delivery systems and the membrane of the cell, leading to the intermingling of both the cell membrane and the liposomes. This then results in liposomal diffusion into target cell membrane lipids, triggering the release of the entrapped therapeutic agents intracellularly.^33,86 The mechanism of lipid exchange is yet to be completely understood, but suggestions has it that this type of mechanism occurs within the cell surface lipid proteins. It is suggested to occur either by a reversible short merging of the monolayer surface of liposomal delivery systems and the cell membrane or through an acyl chain exchange instituted by enzymatic actions between the liposomal delivery systems and the host cell membrane.⁸⁷ Lastly, the mechanism of endocytosis is said to occur intracellularly when the release of entrapped therapeutic agents is triggered by the fusion of endosomes present in the phagocytes of the cell with liposomes which they enveloped. This then leads to the formation of phagosome and is converted through some enzymatic digestive actions into fatty acids.^33,88

Liposomal Delivery Systems Preparation Methods

Liposomal delivery systems are most commonly prepared by employing the “thin film hydration method,” and this method of preparation is carried out by first liquefying the constituting lipids in a chosen organic solvent(s) along with the hydrophobic therapeutic agents. After the liquefaction, the organic solvent(s) used is removed with the help of rotatory evaporator to form a lipid-film, which is then hydrated with the use water or buffer depending on the solvent for preparation (Figure 4). Nevertheless, this method of preparation remains one of various methods used in liposomal delivery system preparation and not the only method used. Some other methods employed for the preparation of liposomal delivery systems therefore includes the “reverse-phase evaporation method,” the “freeze-drying methods,” the “ethanol injection method.”^89,90 There have also been some techniques that have been used such as the sonication, homogenization, membrane extrusion which were added to the preparation list for the main purpose of controlling the liposomal particles distribution as well as their sizes.³³ It is of great importance to emphasize that various liposomal delivery systems obtained from the different preparatory methods comes with varied size distributions as well as fusion to encapsulated therapeutic agents.^90,91 Moreover, the structure and particle sizes of the prepared liposomal delivery systems can be influenced by the variation of the surfactant-to-lipid ratio during the process of fabricating the liposomes. The insertion of surfactant in the bilayers leads to increasing curvature of the bilayers when preparing the MLVs, and this situation can be abated with the help of micro-filters of various sizes (either 0.1 µm, 0.2 µm or 0.45 µm) through filtration; but contrary to this, the sizes of UVs liposomal delivery systems reduces upon addition of surfactant in their little quantities.⁹²

Figure 4.

Thin-film Rehydration method of preparation for liposomal delivery systems.

Direct Comparison of Liposomes and Other Delivery Systems

A summary of the direct comparison of liposomes relative to other nano drug delivery systems can be found in Table 1.

Table 1.

Liposomes vs. Other Drug Delivery Systems

Nano Delivery System	Characteristic Advantages	Disadvantages	References
Liposomes	They possess near perfect biocompatibility They can be used to effectively encapsulate and deliver various kind of drugs. They can be easily multi-functionalized to improve target specific delivery of drugs. They possess relatively low immunogenic possibility.	They can be very unstable within the circulatory system. They require special conditions of storage. Their life cycle is relatively short.	[93–96]
“Solid Lipid Nanoparticles and Nanostructured Lipid Carriers” (SLNs and NLCs)	They possess better stability physically. They possess longer storage life cycle. The possess a more controlled drug release characteristics.	They have limited drug loading capacity. They are less cost effective, and the method of preparation is more complex. They are prone to potential toxicity.	[97–99]
Inorganic Nanoparticles	They possess the ability to convert various forms of energy (for example; light to heat) to cause toxicity to cancer tumor cells.	They could cause toxicity due to possible accumulation. They could have negative impact on the external environment.	[100–102]
Polymer Nanoparticles	They possess specialized chemical compositions. They possess inherent drug release precision. They are versatile, and can be very stable	The process of biodegradation can be complex. They are prone to potential immune response	[99, 103–105]

Liposomal Delivery Systems Used for Docetaxel Delivery Against Prostate Cancer

Nanotechnology avails chemotherapeutic agents with one of the best platforms, whose inherent characteristics could guarantee their specificity, bio-safety, reduced resistance as well as the maximization of their therapeutic activities against prostate cancer. Indeed, the emergence of nano delivery systems have made the possibility of (a) the delivery of large doses of chemotherapeutic agents;¹⁰⁶ (b) co-delivery of two or more chemotherapeutic agents in one formulation;¹⁶ (c) high reduction in bio-toxicity levels by chemotherapeutic agents;¹⁵ and (d) improved target specificity and therapeutic end-point¹⁸ a reality. Therefore, being fully aware of the opportunities and possibilities that the use of liposomal delivery systems presents, scientist have leveraged on these advantages to deliver DTX as a mono-therapeutic agent and in combination with other therapeutic agents against prostate cancer.¹⁰⁷ In this section, the different studies on liposomal delivery systems applied for DTX delivery alone as a mono-therapeutic agent and together with other therapeutic agents as a combinatorial therapy are fully discussed.

Mono-Therapeutic Delivery of Docetaxel Against Prostate Cancer

Generally, in the past few decades, several different chemotherapeutic drugs including DTX have been developed for the therapy of prostate cancer as well as various other kinds of cancer.^108,109 The use of DTX as a mono-therapeutic agent for the treatment of cancer has been reported to record successes at various stages of application such as in vitro, in vivo, preclinical, as well as clinical stages.^39,110,111 The value of mono-therapeutic agents such as DTX can be seen from their prescription benefits in prostate cancer treatment. Notwithstanding, application of these chemotherapeutic agents in their mono-therapeutic forms have been pharmacologically limited by their non-selectivity, high rate of toxicity, random bio-systemic distribution, decreased efficacy, and resistance, amongst others.^112,113 The use of a much better drug delivery system such as nano delivery systems (like liposomes) have been put forward as a possible solution to these pharmacological problems.

In 2016, Ranjan and co-authors embarked on a journey to study how a temperature-sensitive liposomal delivery system used to encapsulate DTX would be distributed (in terms of the concentration of the drug) in a prostate cancer tumor as well as the tumor killing ability.¹¹⁴ According to Ranjan et al, upon analysing the DTX tumor concentration, the prepared DTX liposomes (LTSL) and prepared liposomes plus heat (LTSL + Heat) showed around 4.7-fold higher DTX content (concentration) compared to those in the group of conventional DTX solution and conventional DTX solution plus heat. This was observed from the concentration results which gave DTX tumor concentrations of 6.7 ± 2.8 µg/mL and 9.7 ± 1.8 µg/mL for the groups treated with the prepared liposomes without the presence of heat and in the presence of heat. On the contrary, those in the groups treated with the conventional DTX solution and conventional DTX solution plus heat gave DTX tumor concentrations of 1.85 ± 0.4 µg/mL and 4.8 ± 1.4 µg/mL, respectively.¹¹⁴ Also, from the results of the delay in the growth of the tumor, they observed that the prepared liposomes had no difference in the number of days of growth delay compared to the conventional DTX (17 days of tumor growth each). Interestingly, upon addition of heat, they noticed the group treated with the prepared liposomes plus heat (48.5 days) was able to delay the tumor growth for an additional three days compared to the group treated with the conventional DTX solution plus heat (44.5 days). This results further confirms the potency of their prepared temperature-sensitive liposomes over the conventional DTX solution. They also confirmed the prepared liposomes with or without the presence of heat to induce apoptosis more, as opposed to the conventional DTX solution with or without the presence of heat as seen in higher Caspase-3 upregulation relative to conventional DTX solution. To gain an understanding of the possible effect of the composition of lipid (used for the preparation of delivery systems) in the toxicity of encapsulated drugs, Pereira et al, prepared different liposomes encapsulating DTX with different kinds lipids and evaluated their toxicity against PC-3 prostate cancer cell line in vitro.¹¹⁵ The results of their study showed conventional DTX solution to show high values of viable cells which ranged from 50% to 60% irrespective of the DTX concentration and time of exposure (Figure 5A). On the contrary, the percentages of viable cells were drastically reduced for the various groups treated with the prepared liposomes, irrespective of the compositions of the lipids in the liposomes, but there were also some significant differences in the rate of toxicity amongst the different liposomes prepared (Figure 5B and C). This made the researchers to reach a conclusion that the composition of the lipids in liposomes which affected the efficacy of the toxicity of the encapsulated drug in their study further confirms the importance of making the right choice of lipids when preparing a nano delivery system, especially liposomes. .

Figure 5.

(A) – Results for the cytotoxicity of conventional DTX solution; (B and C) – Results for the cytotoxicity of different lipid compositions for prepared temperature-sensitive liposomes encapsulating DTX. Where *,**,***Represents hourly comparison of “48 hours and 72 hours” (***P < 0.001; for **P < 0.01; for *P < 0.05”), and $$$ represents hourly comparison of “72 hours and 96 hours” ($$$ P < 0.001). “DOPC: 1,2-dioleoyl-sn-glycero-3-phosphocholine,” DSPE-PEG₂₀₀₀: “1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)-2000].”¹¹⁵.

The continuous evolution of science has continued to impact nano delivery systems and this has seen the transition of the preparation of liposomes from the normal conventional liposomes to the liposomes that are tailored to be more specific for target purposes. This involves preparing liposomal delivery systems that could be more passively accumulated in the environment of the prostate cancer tumor. As a result, there is a possibility of increasing cell internalization, ensuring proper interaction of drugs with the action site by the surface functionalization of the prepared liposomes with some ligands. When these ligands interact with the tumor cells receptors, a complex of ligand-receptor is usually formed, triggering endocytosis which precedes the release of drugs intracellularly.¹¹⁶ Thus, liposomes which are prepared to be either stimuli (such as pH) responsive or functionalized with a ligand to help improve their specificity to prostate cancer cells have been evaluated, and several successes recorded when compared to the conventional liposomes as well as conventional delivery system.^117–120

In 2020, Eloy et al, decided to take advantage of passively accumulating liposomal delivery systems at the microenvironment of prostate cancer tumor by preparing a DTX-loaded liposomal delivery system which was functionalized with an antibody cetuximab, and tested against prostate cancer cell lines PC-3 and DU145.¹¹⁷ Cetuximab is an antibody that is known to bind to the “epidermal growth factor receptor” (EGFR), and some cases of prostate cancer are usually associated with the overexpression of the EGFR. Thus, Eloy et al, sort to increase the target specificity of their prepared liposomal delivery system to the prostate cancer cells tumor. The prepared DTX-loaded liposomes were reported to possess an average size of 128.8 ± 2.35 nm; along with a high DTX drug entrapment efficiency of 93.5 ± 2.3%. From the experiment to analyse the ability of the cells to take up the prepared liposomes, it was gathered that the functionalized liposomes uptake was higher (approximately 2-fold) compared to the non-functionalized liposomes in DU145 cell lines (Figure 6A). Toxicity studies using DU145 cell lines also showed that the functionalized DTX-loaded liposomes were significantly more cytotoxic to the cells compared to the non-functionalized liposomes and the solution of the conventional DTX. As observed, functionalized DTX-loaded liposomes showed cell viability of 40.58 ± 6.99% as compared to non-functionalized DTX-loaded liposomes of 53.97 ± 7.43% (Figure 6B and C). However, the same could not be said for the other cell line PC-3, as there was no evident difference in liposomes uptake by these cells. Also, a further evaluation of the toxicity of these prepared DTX-loaded liposomes (functionalized and non-functionalized) in comparison to the solution of a conventional DTX showed no much difference between them using the PC-3 cell lines. The observed results for the PC-3 cell lines showed that the functionalized DTX-loaded liposomes had cell viability of 68.71 ± 2.34% while the non-functionalized DTX-loaded liposomes showed 60.11 ± 10.47% cell viability with 100 nM DTX concentration (Figure 6D and E). Although the numerical values of the results of the solution of the conventional DTX were not stated, it can be seen from the graphical representations (Figure 6A–E) that both the functionalized and non-functionalized DTX-loaded liposomes had better cellular uptake and cell toxicity at the end of the treatment.

Figure 6.

(A) represents the results of cellular uptake of the functionalized (immunoliposomes – [green colour]) liposomes, the non-functionalized liposomes (liposomes – [yellow colour]) and the control (black colour); where * represents the comparison of liposomes to the control group (*p < 0.05, **p < 0.01, and ***p < 0.001) and ^#Represents the direct comparison of liposomes to immunoliposomes (^#p < 0.05 and ^###p < 0.001). (B and C) represents cell viability results for DU145 cell lines for 24 hours and 48hours (Lip-DTX-Ab = functionalized liposomes – [green colour], Lip-DTX = non-functionalized liposomes – [yellow colour], DTX = conventional solution of – [Red colour], and the control (black colour); (D and E) represents cell viability results for PC-3 cell lines for 24 hours and 48hours (Lip-DTX-Ab = functionalized liposomes – [green colour], Lip-DTX = non-functionalized liposomes – [yellow colour], DTX = conventional solution of – [Red colour], and the control (black colour).¹¹⁷

These results come with no surprise to the researchers because PC-3 cells are known to have lesser expression of the EGFR, while the DU145 are known to have a very high expression of the EGFR. This explains why there would be a higher uptake in DU145 compared to PC-3 as the functionalizing antibody (cetuximab) is known to bind strongly to the EGFR. Also, it is of general knowledge that the particle size of liposomes plays a very crucial role in their cellular uptake,³³ and due to the fact that the obtained particle size for the prepared liposomes was small (128.8 ± 2.35 nm), this must have facilitated their uptake intracellularly. Hence more of the liposomes were expected to be taken up by the cells upon the formation of a ligand-receptor complex, and this in-turn led to improved cytotoxicity to the prostate cancer cell line.¹¹⁶

A similar study was carried out recently by Moreira et al. The group of scientists explored the use of a stimuli responsive (pH-responsive) DTX-loaded liposomes as well as a cetuximab functionalized DTX-loaded stimuli responsive (pH-responsive) liposomes compared with a solution of conventional DTX against DU145 and PC-3 cell lines. The study reported an average size for non-functionalized pH-responsive DTX-loaded liposomes to be 107.2 ± 2.9 nm, while that of the functionalized pH-responsive DTX-loaded liposomes was 156.77 ± 1.67 nm.¹¹⁸ DTX entrapment value of 88.65 ± 20.3% was also reported. Just like the results obtained by Eloy et al, in their study,¹¹⁷ the results of the liposomal cellular uptake (cellular uptake at 24 hours of 96.54%) and toxicity (DTX concentration of 100 nM) of the functionalized pH-responsive DTX-loaded liposomes (IC₅₀ of 12.60 ± 2.50 nM) were better than those of the non-functionalized pH-responsive DTX-loaded liposomes (cellular uptake at 24 hours of approximately 90% and IC₅₀ of 28.28 ± 4.60 nM, respectively) for the DU145 cell lines. Also, the toxicity of both functionalised and non-functionalized pH-responsive DTX-loaded liposomes were better than those of the conventional DTX solution (IC₅₀ of 33.55 ± 7.20 nM).¹¹⁸ Ironically, a contrasting result was obtained for the PC-3 cell lines. The results of the conventional DTX solution showed a much better toxicity (IC₅₀ of 55.77 ± 9.21 nM) compared to both functionalized (IC₅₀ of 152.1 ± 25.43 nM) and non-functionalized (IC₅₀ of 65.74 ± 14.61 nM) pH-responsive DTX-loaded liposomes.

This contrasting difference could be as a result of the choice of lipid materials used in the preparation of the liposomal delivery systems, which have been reported to affect the extent of penetration into the membrane of a cell.¹²¹ Although, from the IC₅₀ results of the PC3, it can be seen that the size of the liposomes played a role in the efficacy of the encapsulated DTX, such that a smaller particle size obtained for the non-functionalized liposomes (107.2 ± 2.9 nm) led to better outcome compared to larger size of the functionalized liposomes (156.77 ± 1.67 nm). However, there was less penetration into the PC3 cell owing to the type of materials used in preparing the liposomes, leading to an overall lesser efficacy compared to the conventional delivery system of DTX. Thus a better result was obtained using the DU145 cell lines for the prepared DTX-loaded pH responsive liposomes owing to the surface functionalization of the prepared liposomes.

Generally, liposomes that are functionalized with peptides can be used for selective and efficient delivery of anticancer agents and this includes DTX. These peptides are usually used to target specific receptors present in the tumor, thereby improving the concentration of the drug at the tumor microenvironment.^122,123 Zhang et al, attempted the delivery of DTX using a gastrin-releasing peptide (GRP) functionalized “elastin-like polypeptide (ELP)/liposome” hybrid nano system against PC-3, and DU145 prostate cancer cells. According to the researchers, the DTX was first loaded into the liposomes separately, while the functionalized EPL micelles were also prepared separately. The two different systems were now used to generate a self-assembly hybrid ELP/Liposomes system in the presence of heat.¹²⁰ The study reported the particle sizes to be between 50 to 200 nm but however, they reported low percentage of DTX encapsulation (21%) by the system. Although the group did not carry out so much studies using this prepared functionalized hybrid ELP/Liposomes, they were able to show from the report of the cellular uptake that the prepared hybrid system could be a potential replacement for the conventional DTX. According to their report, PC-3 which is known to exhibit the overexpression of the “gastrin-releasing peptide receptor” (GRPR) was reported to have shown the highest cell fluorescence percentage of 11% in the presence of a “fluorescene-labelled ELP” relative to control which showed 0.4% cell fluorescence. The results of DU145 which showers a lower expression of GRPR was reported to exhibit cell fluorescence percentage of 4.2%. It may catch your attention to know that the prepared hybrid system was also tested on another cell line which is known to be GRPR-negative (293T cell line) which is a kidney cell line, and they found no significant difference between the control cells and those treated with the hybrid nano delivery system.¹²⁰

This phenomenon can be seen as an improved target-site specificity, as the hybrid delivery systems were only able to bind and penetrate the cells that exhibits the expression of GRPR and does not bind to other cells. Also, this could be expected to impact the toxicity of the encapsulated drug (DTX), thereby causing a corresponding drastic reduction of the possible toxicity effects of this drug to normal cells or tissues in the biological environment. Although this functionalized hybrid delivery system may look like the perfect replacement for the conventional delivery system for DTX, it is saddening to say that the research group reported an anticancer efficacy that was less than those of the conventional DTX from the cytotoxicity assay. Zhang et al, believed that this result was due to the poor DTX encapsulation and release into the cell by the prepared delivery system despite the high cellular uptake, and they tried to correlate it to their drug encapsulation and release study which showed 21% was encapsulated and 62% released within 5 hours.¹²⁰ However, non-toxicity of this delivery system could be a trigger for more validating studies to be carried out using this system to prove its potency as an alternative for the delivery of DTX against prostate cancer.

From the literature, it has been reported that there is a possible correlation between the high expression of transferrin receptors and the presence of prostate cancer in males.¹²⁴ A previous study carried out by Kuvibidila et al, detected a highly significant level of “serum transferrin receptor” (sTFR) expression in males that had new diagnosis of prostate cancer, there was no correlation with the stage of the cancer nor tissue inflammation.¹²⁵ Also, a more recent study by Johnson et al, reported the expression of “transferrin receptor 1” in both normal and prostate cancer cells, with the later said to have significantly higher expressions of TFR1 proteins and mRNA.¹²⁶ Therefore, the result of these studies suggest that the possible target of this TFR1 could be a novel approach for the treatment of prostate cancer. Liposomes presents a very good option of functionalization with compounds that can bind to this TFR1 receptor.

An investigation was undertaken by Fernandes et al, which employed the use of transferrin for the functionalization of their prepared DTX-loaded liposomes and tested them against PC-3 prostate cancer cell lines.¹¹⁹ The particle size and entrapment efficiency reported by the study investigators were 79.95 ± 5.57 nm and 69 ± 3.47% for the non-functionalized DTX-loaded liposomes as well as 220.23 ± 3.95 nm and 37 ± 3.15% for the transferrin functionalized DTX-loaded liposomes. Cell viability assay conducted in the study showed a very significant difference (P<0.05) in those treated with the non-functionalized DTX-loaded liposomes (IC₅₀ of 0.19 nM at 72 hours of treatment) relative to both the functionalized DTX-loaded liposomes (IC₅₀ of 26.88 nM at 72 hours of treatment) and the solution of the conventional DTX (IC₅₀ of 34.98 nM at 72 hours of treatment). However, there was no significant difference between the results obtained for the transferrin functionalized DTX-loaded liposomes compared to the conventional DTX solution, but a better toxicity result was recorded.¹¹⁹ Interestingly, these research group also analysed these formulations against a normal prostate cell line (PNT2). Regrettably, the prepared liposomes were reported to be toxic to the normal prostate cell line. The non-functionalized DTX-loaded liposomes (IC₅₀ of 0.23 nM) had the least amount of cell viability, followed by the conventional DTX solution (IC₅₀ of 10.57 nM), before those treated with the transferrin functionalized DTX-loaded liposomes (IC₅₀ of 11.92 nM). The results obtained for the transferrin functionalized liposomes can be seen as a confirmation of the previous study carried out by Johnson et al, which reported the expression of TFR1 in both normal and prostate cancer cells¹²⁶ as seen in the obtained IC₅₀ for the transferrin functionalized DTX-loaded liposomes (IC₅₀ of 11.92 nM and 26.88 nM). From their results one can infer that, the prepared liposomal delivery systems although had better toxicity against the PC-3 cell lines, they lack specificity which could be a true reflection of the choice of materials used in the preparation of the liposomes, and this prepared liposome could cause toxicity in vivo. Notwithstanding, the studies performed are limited in numbers to make such conclusions, and this was acknowledged by the research group. They then suggested further studies such as the “three-dimensional cell models and the xenograft in vivo studies” to be carried out on their prepared liposomal delivery systems.¹¹⁹

The mono-therapeutic use of chemotherapeutic agents like DTX delivered using the conventional drug delivery systems has been known to be recently faced with serious concerns of resistance, toxicity, lack of specificity, decreased anticancer efficacy, amongst others.^112,113 Liposomal delivery systems have been proven to provide a bridge for these short falls of the conventional delivery systems by improving the anticancer efficacy of chemotherapeutic agents through selective delivery of these agents to the cancer cells. From the above studies which prepared liposomes for the mono-therapy of prostate cancer using DTX, it can be seen that all of the studies recorded higher toxicity against the cancer cells compared to the conventional delivery system of DTX, with the exception of one study¹²⁰ which recorded a better anticancer efficacy against PC3 cells compared to the prepared functionalized and non-functionalized liposomes. This peculiarity of this study relative to others could be attributed to the choice of lipids as well as the functioning ligand used in preparing the liposomes,^33,35 which led to a very low encapsulation efficiency of just 21% compare to other studies that recorded 60% encapsulation efficiency and above. This choice of materials for the liposomal system was able to facilitate the uptake of DTX in DU145 but the reverse was observed in PC3 cell line, thereby leading to the reduced anticancer activity against the particular cell line compared to the conventional delivery system.¹²⁰

It is well established that the sizes of liposomes play a crucial role in their possible internalization in cancer cells both in vitro and in vivo. Exploring this advantageous feature of liposomes over conventional delivery systems was a research team led by Klibaim et al. This research team investigated the possible impact of DTX-loaded liposomes (LDTX) sizes on cytotoxicity, cell-uptake, and anti-metastatic efficacy using DU145 cell lines, by employing both two-dimensional (2D) and three-dimensional (3D) spheroid model.¹²⁷ It was gathered from their study that, LDTX with smaller particle sizes (84.18 ± 0.53 nm), which had lesser DTX encapsulation efficiency (47.41 ± 2.92%), exhibited significantly improved cellular uptake, which translated to more improved cytotoxicity compared to the larger sized LDTX (155.17 ± 0.67 nm) with higher encapsulation efficiency (59.36 ± 4.08%) in both 2D and 3D models. Noteworthy, the smaller sized LDTX showed better decrease in the spheroid viability (65.86 ± 3.66%) from the 3D model relative to both large sized LDTX (75.44 ± 1.16%) and bare DTX (78.40 ± 5.29%). Interestingly, the research group also reported an enhanced anti-metastatic phenotype for the LDTX with smaller particle sizes compared to those with larger sizes. Thus, confirming the importance of small liposomes particle sizes in enhancing anticancer efficacy of liposomal nano delivery systems.

Despite the fact that most of the other studies in this category recorded various degrees of successes, it is important to state that most of the studies with the exception of two^114,127 conducted only some basic in vitro studies and did not proceed to in vivo. Generally, the conditions of cells in vitro are different compared to the in vivo biological environment, and this has greatly impacted the translation of different prepared formulation to clinical level. As a result, these prepared liposomal delivery systems are still in the early testing stages, and the validation of the claims of these studies becomes difficult due to the handful of in vitro studies conducted which are not comprehensive enough. Studies like pharmacokinetics in vivo which could help with data generation on the mechanism of absorption, extent of distribution which could determine the possible toxicity or safety in vivo, and the rate of elimination from the circulatory system could be explored, to establish the possible potentials of these systems in clinical trials. Also, one of the very important criteria for a delivery system to go through the clinical trials phases is the stability of such delivery system. A good understanding of the various prepared liposomal delivery system at the molecular level is very critical, as it could give insight to the assembly process; improve the stability; show how these prepared liposomal delivery systems could interact with various biological molecules and membranes in vivo. Having these data would help in the validation of the claims of the effectiveness of these prepared liposomal delivery systems to serve as a potential delivery system for DTX against prostate cancer.

Combination-Therapeutic Delivery of Docetaxel Against Prostate Cancer

Similar to the case of antibiotics where the era of bacteria resistance to antibiotics led to the use of combination therapeutic strategy (such as the use of two antibiotics or antibiotics along with an adjuvant) to overcome resistance,³³ the strategy of combined use of more than one therapeutic agent helps in the reduction of tumor resistance in cancer treatment through synergism.^128,129 One of the well accepted hypothesis of cancer is that, cancer cells are known to be identical at the initial stages of tumor formation, but as mitotic cell division continues, they begin to develop successive mutations and thus, various parts of a tumor may differ in cell configurations. It is for this reason of heterogeneity that the simultaneous use of different therapeutic agents in cancer treatments are beneficial, such that some of the cells may be susceptive to one therapeutic agent while other cells may be susceptible to the other therapeutic agent.¹³⁰ The combination of therapeutic agents have been reported to enhance the efficacy of anticancer drugs in cancer therapy relative to the mono-applications of these therapeutic agents.^129,131,132 Unfortunately, just as the saying goes that the use of more therapeutic agents translates to possible greater risks of therapeutic side or adverse effects, the same applies in this case. This means there are possibilities of greater side effects with the application of combined therapeutic agents compared to mono-therapeutic agents in cancer treatment.¹²⁹ Pathetically, when these side effects are developed, it may be a mysterious process trying to unravel which of the therapeutic agents that is responsible for the side effects experienced, and when such side effects are severe, the therapy may be discontinued.¹²⁹ Additionally, the use of more than one therapeutic agent also translate to higher possibility of drug-drug interaction, which can also lead to adverse effects.¹²⁹ To address these abovementioned challenges especially in the treatment of prostate cancer, liposomal delivery systems which are known to advantageously deliver hydrophilic and lipophilic drugs simultaneously, have been proposed as the possible solution.²⁸ The use of liposomes for combination-therapeutic strategy for DTX combined with some other therapeutic agents such as chemotherapeutic drugs, peptides, amongst others are discussed herein below.

In 2020, Rushworth and co-authors aspired to invent novel treatment strategy for the treatment of prostate cancer that are chemo-resistant by improving the therapeutic efficacy of DTX and thereby reducing resistance. Rushworth et al, then prepared a liposomal delivery system (transferrin [TF] functionalized and non-functionalized) for the combined delivery of DTX and an anthelmintic agent mebendazole (MBZ) and evaluated it against LNCaP and PC3M-Luc-G5 cell lines¹³³ The developed liposomes were reported to exhibit different particle sizes and entrapment efficiency for the different therapeutic agents (singly and combined), and these includes (a) liposomes encapsulating MBZ [LIPO MBZ] 118.4 ± 0.47 nm and 44%; (b) liposomes encapsulating DTX [LIPO DOC] 73.4 ± 0.17 nm and 80.4%; (c) liposomes encapsulating DTX and MBZ [DOC + MBZ] 133.2 ± 0.71 nm and 43% (MBZ), 83.4% (DOC); and TF functionalized liposomes encapsulating DTX and MBZ [TF DOC + MBZ] 137.8 ± 0.53 nm and 44.3% (MBZ), 83.7% (DOC). The in vitro anti-proliferation efficacy against LNCaP and PC3M-Luc-G5 cell lines showed the TF functionalized and non-functionalized liposomes which encapsulated MBZ and DTX combined (TF MOC + MBZ; DOC + MBZ), to be more potent compared to their individual liposomes preparation (Figure 7A and B). Interestingly, they noted a much higher anti-proliferating efficacy exhibited by the TF functionalized liposomes of combined DTX and MBZ against the LNCaP cell lines (Figure 7B). Rushworth and co-authors further explored the efficacy of their prepared liposomes in vivo using the PC3M-Luc-G5 cell lines. The researchers subcutaneously injected the PC3M-Luc-G5 cells in male BALB/c mice that were immune-deficient, and treated the animals via the same route of administration. An obliteration of tumor growth was observed during the period of treatment (1–9 days) for the group treated with both the TF functionalized and non-functionalized liposomes which contained DTX and MBZ combined. This observation was in contrast with what was observed for the groups treated with liposomes containing the individual drugs singly (Figure 7C). Moreover, classification of the treated tumors into different categories such as: (a) the progressive; (b) stable; and (c) partially regressed, saw the tumors belonging to the group treated with TF functionalized and non-functionalized liposomes containing combined DTX and MBZ being classified as 90% and 50% partially regressed (Figure 7D). This was relative to most of the tumors treated using other treatment options (blank liposomes; liposomes which contains DTX alone and MBZ alone) and the untreated group, which were categorized as being progressive (Figure 7D). Also, it is intriguing to mention that, liposomes containing TF MOC + MBZ; DOC + MBZ were reported to significantly extend (for 6.5 and 5 days) the “progression-free survival” (< 20% tumor size increase from the original volume at day one) relative to liposomes which contained DTX [DOC] alone (0.038); MBZ alone [0.007]; blank liposomes; and untreated group (0.0007) (Figure 7E). Recently, a study group under the leadership of Zhang et al, also explored the possibility of developing a novel PEGylated liposomal delivery system for the co-delivery of Resveratrol (Res) and DTX against PC-3 prostate cancer cell lines. The prepared liposomes which includes Doc-LPs (DTX); Res-LPs; blank liposomes (Blank-Lps) and Doc/Res-LPs showed size and entrapment efficiency of 92.21 ± 2.08 nm and 80.85 ± 0.24%; 103.39 ± 6.89 nm and 89.11 ± 0.74%; 82.11 ± 2.28 nm; 99.76 ± 3.14 nm and 81.52 ± 3.13% [Doc] / 85.27 ± 1.74% [Res], respectively.¹³⁴ Cellular uptake analysis of the prepared liposomes (Doc-LPs or Doc/Res-LPs) revealed a statistically significant higher uptake of DTX [Doc] in PC-3 cells (with uptake of 59.44 ± 0.48% for Doc-LPs and 73.58 ± 2.79% for Doc/Res), relative to the conventional DTX solution [Doc-Sol with uptake of 18.36 ± 0.40%] (Figure 8A). Likewise, was there a statistically different increase in the percentage uptake of Res in Doc/Res-LPs (22.90 ± 0.85%) and Res-LPs (19.51 ± 0.91%) compared to Res-Sol (6.63 ± 0.25%).¹³⁴ Zhang et al, then proceeded to conduct various cytotoxicity experiments (for 4hours) with the prepared liposomes (Blank-LPs; Doc-LPs; Res-LPs; and Doc/Res-LPs) in comparison with the solution (Doc-Sol; Res-Sol; and Doc/Res-Sol) of the conventional drugs. The result of these cytotoxicity experiments revealed a significantly higher anti-proliferative activity by the Doc/Res-LPs than those of Doc/Res-Sol and Doc-Sol, which supports synergy in co-treatment, while the Blank-LPs barely inhibited the growth of the cells (Figure 8B). Also, a significant increase in the enzymatic activity of caspase – 3 in PC-3 treated cells with Doc/Res-LPs and Doc/Res-Sol compared to other treatments, with the former reported to have a higher effect than the later (Figure 8C). After obtaining these results, it became invaluable for the research group to take it further by performing an in vivo anti-tumor studies using the same cell line (PC-3). They gathered that, although there was a significant decrease in the volume and weight of the tumor by the group treated with Doc-LPs and Doc/Res-LPs relative to the control group treated with saline, the same could not be said when compared to Doc-Sol and Doc/Res-Sol (Figure 8D–F). Against all odds, it was noticed that there were no body weight changes in the mice belonging to the group treated with Doc-LPs and Doc/Res-LPs with no fatality recorded, whereas those treated with Doc-Sol and Doc/Res-Sol lost some significant amount of body weight as well as total fatality between day 8 and 9 (Figure 8G and H). Lesser toxicity to organs was also reported for the liposomes compared to the conventional DTX in vivo.¹³⁴ Thus leading to an incomplete treatment cycle for these groups. It can therefore be gathered from the above two studies that, co-delivery of DTX with another chemotherapeutic such as MBZ and Res through the use of liposomal delivery systems could help to improve the target-specificity of DTX against prostate cancer, and in-turn decrease the resistance posed to this chemotherapeutic agent by these cancer cells. However, there are still a lot of grounds to be covered in terms of the number of studies evaluated in these two studies, as the in vivo of one of these studies, communicated no much difference between the prepared liposomal systems (alone and combined) compared to the solution of conventional delivery system of DTX.¹³⁴ Therefore, more intensive studies are encouraged.

Figure 7.

Graphical representation of (A) – Anti-proliferation of prepared liposomes against PC3M-Luc-G5 cell lines (where *p < 0.05, **p < 0.001, ***p < 0.0001); (B) – Anti-proliferation of prepared liposomes against LNCaP cell lines (where *p < 0.05, **p < 0.001, ***p < 0.0001); (C) – Relative volumes of the tumor treated in vivo; (D) – The tumor’s response to various treatment in vivo; and (E) – Progression – free rate of survival (where *p < 0.05).¹³³

Figure 8.

Diagrammatical representations of (A) – The percentage uptake of Doc and Res in the treated cancer cell PC3 with various prepared formulations; (B) – The percentage of PC3 cancer cell viability when treated with the different formulations in vitro; (C) – The enzymatic effect of Caspase – 3 on the PC3 cancer cells treated with the different formulations; (D) – The tumors from the in vivo study; (E) – The changes in the volumes of the tumors; (F) – The changes in the weight of the tumors; (G) – The changes in the body weight of the treated animals; (H) – the changes in the percentage survival of the PC-3 tumor carrying animals for the period of the treatment (study). *(P < 0.05) and **(P < 0.01) indicates statistical differences relative to the Doc-Sol group for figure (A); *(P < 0.05) and **(P < 0.01) indicates statistical differences betwixt Doc/Res-LPs and Doc/Res-Sol group while ^#(P < 0.05) and ^##(P < 0.01) indicates statistical differences betwixt Doc/Res-LPs and Doc-Sol group for figure (B); *(P < 0.05) and **(P < 0.01) indicates statistical differences relative to the control while ^#(P < 0.05) and ^##(P < 0.01) indicates statistical differences relative to the Doc-Sol group for figures (C); *(P < 0.05) and **(P < 0.01) indicates statistical differences relative to the control while ^#(P < 0.05) and ^##(P < 0.01) indicates statistical differences relative to the Doc/Res-LPs for figures (E and F), while red box in figure (E) amplifies the crowed graph area of origin for easy understanding, and the ns indicates non-significant difference between the blank liposomes and the control for figure (F).¹³⁴

Inorganic nanoparticles such as “gold nano-rods” (GNRs) have been proven to be effective against cancer through the use photo-thermal treatment strategy, as well as a corresponding reduced toxicity to normal tissues or healthy organs.^135,136 Notwithstanding, GNRs have been reported to possess poor stability and cellular-uptake when present in a biological environment, which has led to their limited use as anticancer agents.¹³⁷ It has been reported that a combination of GNRs and chemotherapeutic agents against cancer can help improve cancer therapy, especially when this delivery is mediated through the use of nano delivery systems like liposomes.¹³⁵ Hence, just like the co-delivery of other chemotherapeutics alongside DTX using different types of liposomal delivery systems, the co-delivery of inorganic metallic nanoparticles like GNRs with DTX has also been explored.¹³⁸ In 2017, Hua and fellow authors investigated ability of peptide and polyethylene glycol functionalized liposomes (GNRs/DocL and GNRs/DocL-R; [R – peptide, Doc – DTX]) used to encapsulate GNRs and DTX to improve prostate cancer therapy both in vitro and in vivo.¹³⁸ According to them, the prepared liposomes had average size of 163.15 ± 1.83 nm as well as a loading efficiency (entrapment efficiency) of 98.45% for DTX. Upon examining the cell inhibitory efficacy in vitro against PC-3, they found the conventional DTX solution to inhibit the growth of the cell slowly in a concentration dependent manner as opposed to the prepared liposomal delivery systems, with or without a laser within 24 hours of treatment (Figure 9A). They also noted a significant inhibition of cell growth of approximately 100% (DTX concentration of 80 µg/mL) upon extension of the time of exposure of these cells to the prepared liposomes with laser (synergism), which was not the case for the conventional DTX solution (Figure 9B). The induction of anti-apoptotic effect by cancer cells as well as the reduction of excess ROS present intracellularly (Note: excess intracellular ROS is usually induced by DTX, preceding apoptosis), are known to be amongst the mechanisms by which resistance to DTX is posed by prostate cancer cells.^47,57,58,64 Apoptotic effect and excess intracellular ROS inducement was therefore analysed, and the results showed conventional DTX solution to cause a cell necrosis, early apoptosis and late apoptosis of 0.2%, 16.8%, 6.4%; and low amount of excess ROS intracellularly, respectively. Interestingly, the prepared liposomes plus laser induced cell necrosis, early apoptosis and late apoptosis of 0.1%, 50.2%, 2.7%; and higher amount of excess ROS intracellularly (Figure 9C and D). Also, to confirm the phase of cell growth inhibition, the cell cycle of the prostate cancer cells (treated and untreated) were analysed. Conventional DTX solution was seen to increase the G₂/M cell percent from 11.3 ± 0.6% in the untreated group to 34.2 ± 1.2%; while the two prepared liposomes (liposomes and liposomes plus laser) caused a significant increase to 67.4 ± 1.8% and 76.8 ± 1.6% respectively. In the G₀/M phase where DNA duplication is usually carried out in preparation for mitotic cell division, the untreated group was observed to have approximately half the total percentage of cells; DTX treated group had approximately 34%; liposomes group had 20.0 ± 0.9% and liposomes plus laser group had 5.7 ± 0.4% (Figure 9E and F). Thus showing the superiority of prostate cancer cells inhibition in vitro by the prepared liposomes over the conventional DTX solution.¹³⁸ Furthermore, the antitumor efficacy of the prepared liposomes was also compared to that of the conventional DTX solution as well as a group of untreated nude mice bearing PC-3 tumor. It was gathered that the prepared liposomes exhibited significant tumor reduction differences in relation to the tumor volume (Figure 9G); tumor weight (Figure 9H) and tumor size (Figure 9I) compared to the conventional DTX solution and the untreated group. They also noted that the weight of the animals (Figure 9J) in the groups treated with liposomal delivery systems were highly maintained as opposed to the group treated with conventional DTX solution which recorded the highest body weight loss through the course of the experiment. These in vivo results further confirmed the results obtained in vitro, thereby amplifying the superiority of co-delivery of GNRs and DTX using a functionalized liposomal delivery system over the conventional DTX against prostate cancer. .

Figure 9.

Where (A) – Cell inhibition (24 hours); (B) – Cell inhibition (80 µg/mL of DTX) (12–72 hours); (C) – Cell apoptosis; (D) – ROS control; (E and F) – Cell cycle analysis; (G) – Tumor volume; (H) – Tumor weight; (I) – Tumor sizes; (J) – Animal body weight. “⁺GNRs/DocL-R vs. GNRs/DocL-R +laser, (P<0.05); *GNRs/DocL + laser vs. GNRs/DocL-R + laser, (P<0.01); ^#GNRs/DocL vs. GNRs/DocL + laser, (P<0.05).”¹³⁸.

Apart from the above discussed materials which have been co-delivered with DTX using liposomal delivery systems, “Small Interfering Ribonucleic Acid (siRNA)” has also been explored by some group of scientist as discussed below. In 2019, Zhang along with some collaborators prepared liposomal delivery systems which encapsulated siRNA and DTX, and was analysed against prostate cancer cell lines to show its efficacy over conventional DTX.¹³⁹ The researchers mentioned that they obtained an average particle size of 39.7 nm, while the entrapment efficiency for siRNA and DTX were 82.4% and 83.8% each. It was also put forward from their cytotoxicity assay results that only an IC₅₀ concentration of 0.15 µg/mL of the prepared siRNA and DTX-loaded liposomes was required to reduce the percentage of proliferating treated PC-3 cells to 50%. Nevertheless, a very much higher IC₅₀ concentration of 0.27 µg/mL for the solution containing siRNA and conventional DTX as well as 0.32 µg/mL of conventional DTX solution alone was needed for the same purpose.¹³⁹ After 24 hours of treatment of the cells, the cell viability of the groups which includes liposomes (siRNA + DTX); solution of siRNA + DTX; solution of DTX; solution of siRNA; and the untreated group which served as control were reported to be 42%, 63%, 84%, 95%, and 100%, respectively (Figure 10A). Analysing the apoptotic effect of the various treatment groups showed the liposomes treated group had a total of 66.4% cell apoptosis rate, while the groups treated with solution containing siRNA + DTX; DTX; siRNA and the untreated group exhibited total apoptosis of 37.1%, 17.7%, 2.5%, and 1.9% (Figure 10B). These results corresponded to G₂/M cell arrest of 30%, 24.1%, 23.2%, 19.7%, and 18% from the cell cycle analysis which was also carried out by the research group (Figure 10C). Also, the in vivo study conducted to establish the efficacy of the prepared liposomal delivery system against PC-3 tumor showed the prepared liposomes to be more effective reducing the volume (Figure 10D) and size (Figure 9E) of the tumor, as well as improving the rate of survival of the animals (Figure 10F), relative to other groups. It was fascinating to see that Zhang et al, reported no toxicity in the animals from various groups (treated and untreated) with exception to the group treated with conventional DTX solution, which was reported to be toxic to the heart of the animals,¹³⁹ This goes a long way to tell more about the integrity of the prepared liposomal delivery system in connection with their ability to overcome toxicity posed by DTX to normal cells and tissues in the body.

Figure 10.

(A) – percentage cell viability; (B) – percentage apoptotic effect [EA – early apoptosis, LA – late apoptosis, and TA – total apoptosis] (where *p < 0.05); (C) – percentage cell circle; (D) – the volume of the tumor (where *p < 0.05, **p < 0.01); (E) – the sizes of the tumor; and (F) – the percentage rate of animal survival.¹³⁹

Furthermore, there have been efforts made by researchers to use both low-intensity-ultrasound, sonosensitizers and chemotherapeutic agents as a combination therapy for the treatment of cancer, and this combination is referred to as “sonodynamic therapy” (SDT).¹⁴⁰ This SDT induces toxicity against cancer by generating ROS through the combination of low-intensity-ultrasound and sonosensitizers.¹⁴⁰ Taking advantage of this SDT, Dai coordinated a research group that prepared a sonosensitive (using IR780 as sonosensitizer) phase-changing liposomes for the co-delivery of anti-prostate-specific membrane antigen (anti-PSMA) and DTX against prostate cancer.¹⁴¹ According to their result, the prepared liposomes had particle size of approximately 226.4 ± 48.2nm, with drug entrapment efficiency of 17.77%. The in vitro cytotoxicity assay conducted using C4-2 prostate cancer cell lines revealed that the prepared liposomes had enhanced toxicity in the presence of “low-intensity-focused-ultrasound,” with highest apoptosis rate of 30.94%. This enhanced anticancer activity was owed to the increased permeability of the cell membrane by the prepared liposomes which led to the accumulation of the liposomes inside the cell, despite the low entrapment efficiency of the prepared liposomes. The research group went further to conduct in vivo studies and the results obtained correlated with those obtained in vitro. The tumor volume was reported to have shown only 15.03% increase as opposed to the control group which showed 597.15% tumor volume increase. The obtained results led to the conclusion by the authors that, the prepared sonosensitive phase-changing liposomes has a possible clinical potential, and could help in the effective visual treatment prostate cancer.¹⁴¹ In 2025, Dai et al, embarked on a study to improve the therapeutic efficacy of DTX-loaded liposomes, and thus, prepared a versatile liposomal formulation loaded with both DTX and nucleic acid for combination therapy against PC-3 prostate cancer cell lines both in vitro and in vivo.¹⁴² The prepared liposomes (MDS@LA) with reported particle size between 160–190 nm, were regarded as versatile due to the presence of “mesoporous polydopamine” (MPDA), and target-specific ligands such as aptama, siRNA. From the results of the cellular uptake, it was reported that the presence of the targeting ligand Aptamer led to improved cellular uptake relative to the bare DTX in PC-3 cancer cells in vitro. Remarkably, the same report was given for the in vivo studies, where the prepared liposomes showed better tumor reduction capability compared to the bare DTX.¹⁴² This amplifies the potential of a multifunctionalized liposomes relative to the conventional delivery system.

Liposomal delivery systems are known to possess a characteristic lipophilic bilayers and a hydrophilic internal core, allowing them to be used for combinational therapy of both lipophilic and hydrophilic drugs.³³ Regrettably, this advantageous characteristics of liposomes are yet to be well explored, and this can be observed with the few studies recorded in the monotherapy and combination therapy section above. From the studies above which are summarized in Table 2 below, from the cytotoxicity assays through the cell cycle assays and the apoptotic effect in vitro, to the various studies in vivo, for the various liposomal delivery systems prepared and studied, showed a predominantly better results compared to the conventional DTX solution and the physical mixtures of DTX and the various chemotherapeutic agents. This can be seen as a step in the right path in relation to improving the efficacy of DTX and also reducing the resistance posed to DTX by prostate cancer cells and tumors. Nevertheless, there are still a lot of rooms for improvement in terms of the quality of studies to be done to ascertain the efficacies of these delivery systems such as in vivo toxicity to normal cells, organs and tissues, which was only reported by a few studies,^134,139 as well as pharmacokinetic studies amongst others.

Table 2.

Co-Delivery of DTX and Other Chemotherapeutic Agents

Liposomal Constituents	Type of Co-Delivery Agent	Co-Delivery Agent	Design of Study	Type of Cell Lines	Results	References
Transferrin; DSPE-PEG2000-Mal; Chol; and DOPC	Chemotherapeutic Drug	Mebendazole	In vitro and in vivo	LNCaP and PC3M-Luc-G5	Superior anticancer efficacy was recorded in both in vitro and in vivo studies relative to conventional DTX solution.	[133]
Chol and DSPE-PEG2000	Chemotherapeutic Drug	Resveratrol	In vitro and in vivo	PC-3	The prepared liposomes showed better anticancer effect compared to conventional DTX in vitro but no significant difference in vivo.	[134]
SPC; HSPC; Chol; GNRs; DSPE-PEG2000-Mal; and RLT peptide	Inorganic Agent	Gold nano rods (GNRs)	In vitro and in vivo	PC-3	The prepared liposomes showed better anticancer effect compared to conventional DTX both in vitro and in vivo.	[138]
DOPA; Calcium phosphate; DSPE-PEG2000-RGD (Arginine-Glycine-Aspartic acud)	Small Interfering Ribonucleic Acid	SiRNA	In vitro and in vivo	PC-3	The prepared liposomes showed better anticancer effect compared to conventional DTX both in vitro and in vivo.	[139]
Chol, DSPE-PEG2000, DPPC, perfluoropentane (PFP), and IR780 (sonosensitizer)	Antigen	Anti-PSMA	In vitro and in vivo	C4-2	The prepared liposomes showed better anticancer effect compared to control both in vitro and in vivo.	[141]
EPC; HSPC; Chol; DSPE-PEG2000-Mal; MPDA; Aptama;	Small Interfering Ribonucleic Acid	SiRNA	In vitro and in vivo	PC-3	The prepared liposomes showed better anticancer effect relative to the bare DTX both in vitro and in vivo.	[142]

Recently, there have been some innovative studies which have tried to synthesize novel lipids for the preparation of liposomes for the delivery of anticancer agents.¹⁴³ According to a meta-analysis report which was put forward by Petersen et al, it was gathered that there was indifference in terms of efficacy of the “first generation liposomal drugs” and the chemotherapy delivered using the conventional delivery systems.¹⁴⁴ From the studies above, the materials used for prepared liposomes were lacking innovative materials in the composition of the liposomes, with exception to the functionalizing ligands (Tables 2 and 3). Researchers have recently dedicated their efforts to the preparation of novel liposomal delivery systems for the delivery of chemotherapeutic agents, to help improve the potential safety in vivo, as well as their anticancer efficacy. This has been achieved with the use of innovative materials, which has led to the emergence of many novel liposomal drugs recently.¹⁴³ Some of these new liposomal delivery systems are long circulating, immune-responsive, cationic, sensitive to the immediate biological environment of the tumor, and thus have helped to improve the target-specificity, time of circulation (half-life), biological safety, and ultimately the efficacy of the prepared liposomal delivery systems.^{143,145–147}

Table 3.

Mono-Therapeutic Delivery of DTX Against Prostate Cancer

Liposomal Constituents	Liposome Type	Functionalized Ligand	Design of Study	Type of Cell Lines	Results	References
DPPC; DSPE-PEG-2000; MSPC; and DSPG	Temperature-sensitive conventional liposomes	Not Applicable	In vivo	PC-3	Significantly higher concentration of DTX in the tumor were recorded for the prepared liposomes both with or without the presence of heat relative to those obtained from conventional DTX solution with or without the presence of heat against PC-3 cell lines. No difference in delay of tumor growth for conventional DTX solution and prepared liposomes without the presence of heat. Liposomes with the presence of heat delayed tumor growth for extra three days compared to conventional DTX solution in the presence of heat.	[114]
Chol; DOPC and DSPE-PEG −2000	Conventional liposomal delivery systems	Not Applicable	In vitro	PC-3	Significantly higher cellular toxicity was recorded for the different lipid composition types of liposomes prepared relative to conventional DTX solution against PC-3 cell lines.	[115]
Chol; SPC; DSPE-PEG-Maleimide; and Cetuximab	Functionalized liposomal delivery system	Cetuximab antibody	In vitro	DU145 and PC-3	Higher cellular uptake was recorded for functionalized liposomes relative to non-functionalized liposomes and control against prostate cancer cells evaluated. Significantly higher cellular toxicity was recorded for functionalized liposomes relative to non-functionalized liposomes and conventional DTX solution against DU145 cell lines. Cellular toxicity of functionalized liposomes, non-functionalized liposomes and conventional DTX solution against PC-3 showed no remarkable difference.	[117]
DOPE; DSPE-PEG −2000; CHEMS and Cetuximab	Functionalized pH-responsive liposomal delivery system	Cetuximab antibody	In vitro	DU145 and PC-3	Higher cellular uptake was recorded for functionalized liposomes relative to non-functionalized liposomes. Significantly higher cellular toxicity was recorded for functionalized liposomes relative to non-functionalized liposomes and conventional DTX solution against DU145 cell lines. Conventional DTX showed higher cellular toxicity of relative to those treated with the functionalized liposomes and non-functionalized liposomes against PC-3 cell lines.	[118]
HSPC/MPEG-DSPE; lecithin; Chol; DSPC; DOPC; ELP; GRP	Functionalized hybrid ELP/liposomal delivery system	GRP	In vitro	DU145 and PC-3	Higher cellular uptake was recorded against prostate cancer cells with expression of GRPR. No cellular uptake for non-prostate cancer cell lines that lacks expression of GRPR. Reduced efficacy against PC-3 cells compared to conventional DTX solution.	[120]
Chol; SPC; DSPE-PEG-Maleimide; and Transferrin	Functionalized liposomal delivery system	Transferrin antibody	In vitro	PC-3	Significantly higher cellular toxicity was recorded for non-functionalized liposomes relative to functionalized liposomes and conventional DTX solution against tested cell lines. No notable difference in efficacy for the functionalized liposomes relative to the conventional DTX solution against tested cell lines.	[119]
SPC; Chol; and PE-PEG	Not Applicable	Not Applicable	In vitro	DU145	Significantly improved cytotoxicity, cellular uptake, and anti-metastatic activity in the small sized LDTX compared to both large sized LDTX and bare DTX.	[127]

The preparation methods, choice of materials (cost inclusive) as well as the models used for the various experiments plays a very crucial role in the possible outcome of the prepared liposomal delivery system. There have been documented evidences that most of these prepared liposomes which were shown to have small particle sizes, good entrapment efficiency, as well as improved cellular uptake (in vitro) and good anticancer efficacy (in vivo) using the mouse models fails to move to different stages in clinical trials.^148,149 From the studies reported (Tables 2 and 3), most of the in vivo studies made use of the xenograft models for their preclinical animal studies, whereby the cancer cells were subcutaneously inoculated into nude nice that are immunocompromised. This gives the studies the main specific limitation of the xenograft model, which is the inability to appraise the possible effect of the immune system response to either the grafted tumor or the prepared liposomes used for the treatment. The use of allograft models, which are not immune-compromised could be a better alternative to this xenograft model, and could give a much refined outcome for the prepared liposomes.^148,150 Thus, considering the cost of materials, there is need for the validation of the prepared liposomes (Tables 2 and 3) that have been functionalized with innovative materials to ensure reproducibility during the scale-up process, retaining its core characteristics such as the particle size of the prepared liposomes, their drug encapsulation efficiencies, stability, and the eventual translation of the in vivo efficacy to clinical trials stages.

Lastly, the toxicity profile of these liposomes is of great importance as it would go a long way in determining the possible transition of these prepared liposomes from one preclinical or clinical stage to the other. Liposomal DTX has been previously shown to give rise to several additional toxicities in a phase one clinical study that was conducted previously by Deeken et al, some years ago.¹⁵¹ From their study, they discovered that many of the patients experienced alopecia; edema; dtspnea; rash; amongst other toxicities, which were secondary to the primary neutropenia experienced.¹⁵¹ This may have stemmed from their choice of materials used from the liposomes preparation, which influenced the distribution and release of the encapsulated DTX in the biological environment of the host patient. Thus, optimization of the prepared liposomes with regards to choice of materials, method of preparation as well as the use of proper and validated models for evaluation of liposomes in vivo would facilitate their entrance transition to clinical trials, and give better efficacy with reduced toxicity during clinical trials. This could potentiate the futuristic approval for their application as a delivery system for the delivery of DTX against prostate cancer.

Clinical Transformation Challenges of Liposomes

Generally, the clinical potentials of liposomes have been estimated to be very exceptional, when it comes to their ability to delivery drugs, and this has accorded them a very significant attention from the scientific community as well as the pharmaceutical industry. Currently, there are a few liposomes for different anticancer agents that have made it to the clinical trials stages, and these include the “RGD-decorated nanoliposomes arsenic trioxide and curcumin,”¹⁵² “MethodsPoly (2-ethyl-2-oxazoline)-dioleoyl phosphatidylethanolamine-nanoliposomes,”¹⁵³ and “Liposome-curcumin and resveratrol.”¹⁵⁴ Despite the great potentials exhibited by these liposomes, there remain numerous challenges that has persistently posed obstacles to their possible translation from just basic studies carried out in the laboratories to a possible well founded clinically effective application. A good example of these challenges is the fact that, an antitumor drug carrying liposomes can experience instability due to the inherent structural bilayers formed by the phospholipids, and this could result in hydrolysis, which could ultimately affect the extent of bioavailability of the drug owing to leakage.¹⁰³ Meanwhile, there remain the possibility of these liposomes causing hepatotoxicity, and this amplifies the need for further studies to be conducted on these liposomes before they can proceed to clinical trials. Importantly, the mechanisms of release of these encapsulated drugs in vivo, would be an asset when trying to design the liposomes, as it could give some valuable information about possible interactions or toxicity of the prepared liposomes, which ultimately affects their clinical translation.¹⁵⁵

Conclusion and Future Perspectives

The burden of resistance and reduced toxicity of DTX to prostate cancer cells still continues to plague and flaw the efforts medical professionals for the therapy of prostate cancer worldwide. Nano delivery systems such as liposomes have proved to be a possible solution to these problems either in the form of mono-therapeutic or combined therapy of DTX with other chemotherapeutic agents, with various studies recording great success in this regard. Generally, the ability of liposomes to successfully encapsulate drugs within their bilayers have been a strong argument for their necessitated clinical applications, and this has led to the improvement of the therapeutic efficacy of the encapsulated drugs through alteration of pharmacodynamics and pharmacokinetic properties.⁹³ Additionally, the ability of these liposomes to modulate the behaviour of the encapsulated drugs in vivo, help to reduce the toxicity of the drug against the normal cells in the body during clinical trials, and this feature remains a crucial factor to be considered when preparing these liposomes. However, there are potential risk involved in the maintenance of the physicochemical as well as biological characteristics of these liposomes during the process of sterilization of methods or scale-up, such that a slight change in material could lead to stability problems, and a drastic variation in the pharmacodynamics or pharmacokinetics of the encapsulated drug. Therefore, experiments to validate these prepared liposomes would be of great importance, and more novel studies should be carried out, owing to the selected few that has been evaluated in the past decade, so as to widen the pool of liposomal delivery systems for DTX delivery against prostate cancer. To achieve the aim of improving these liposomes if they are to translate into clinical trials, it is important to consider developing new target specific ligands that can bind to both already established biomarkers, and new or yet to be established possible biomarkers.^103,156 Also, owing to the presence of diverse kinds of barriers in the internal biological environment, the utilization of innovative smart materials for the preparation of liposomes that are stimuli sensitive to both temperature, enzyme, pH or light, design of some potential hybrid nanoparticles, as well as employing the right animal models for the preclinical studies could be of great benefit in improving their therapeutic efficacy when translated to clinical trials. This could in-turn lead to a futuristic end to the resistance and low therapeutic efficacy experienced with the use of DTX in the therapy of prostate cancer clinically. Thus validating their potential as possible delivery systems for DTX against prostate cancer.

Funding Statement

This study was funded by the Faculty of Health Sciences of the University of the Free State, (Grant No: 1047 B4320).

AI Statement

During the preparation of this work, the authors did not make use of any artificial intelligence (AI) tool for either the writing or curation of data.

Disclosure

The authors declare no conflicts of interest in this work.