Introduction

Over the past 70 years, significant advancements have been made in drug delivery technology. A crucial development in modern pharmacy is the invention and implementation of carrier-based drug delivery systems, specifically those using nanometer-sized particles. These carriers offer several advantages over traditional drug delivery systems, effectively addressing various challenges in disease treatment.1,2 Nanocarriers are vehicles for drugs, acting as a protective shield within the body. They shield drugs from degradation in the physiological environment throughout the pre-release process, prolonging the drug’s half-life and enabling targeted delivery. Targeted delivery drug carriers, including liposomes, lipid-based nanoparticles, protein-based nanoparticles, polymer nanoparticles, and inorganic nanoparticles, can deliver drugs to designated sites, enhancing drug efficacy, improving drug bioavailability, minimizing damage to normal tissues and cells, and reducing side effects.1,3 Traditional systemic drug delivery methods have several limitations, such as low drug-specific bioavailability, inadequate drug concentration at the target site, and significant adverse effects. As a response to these challenges, targeted therapies have emerged.3,4 Moreover, the invention and advancement of targeted delivery drug carriers hold great significance in treating diseases that require precise drug release, such as cancer. The aforementioned targeted delivery drug carriers encompass various types that have gained considerable attention and research, including nanogels, micro/nanomotors, microbubbles, polysaccharide, biomimetic nanoparticles, extracellular vesicles, yeast, and viral vectors.5–10 With continuous advancements in passive targeting, active targeting, and other targeting methods, the diversity of targeted delivery drug carriers continues to expand.

Many delivery carriers with targeting capabilities have been mentioned; however, how many nanocarriers have made it to market? We have compiled a table of the nanocarriers approved by the Food and Drug Administration (FDA) (Table 1), including those with “Prescription” and “Discontinued” status. Of these, four of the liposome-based drugs have a “Discontinued” status; only one of the micelle-based drugs was approved but is now also with a “Discontinued” status, and one of the iron oxide nanoparticles-based drugs has a “Discontinued” status. The FDA has approved 13 nanocarriers, six of which are with a “discontinued” status. The discontinuation of a marketed drug is complex and includes (1) problems with the safety, efficacy, or controllable quality of the drugs; (2) emergence of new, more efficacious, decrease in the market share; (3) owing to the control by the relevant departments, the supply of raw materials is difficult because of the environmental pollution, hindering the production; (4) low end-demand and small market share of the disease cured by the drugs; (5) increase in costs of production. In addition to production, market, policy, and other external factors, the efficacy and safety of the drug need extra attention. In the field of nanomedicines, a small proportion of the research focuses on using nanocarriers to deliver drugs. In the field of nanocarrier delivery, researchers focus more on the nature of the loaded drug than the nature of the blank nanocarriers. Additionally, there is a tendency to overly concentrate on the design of the nanocarriers themselves. Most pharmaceutical researchers design the carrier in a very subtle way and modify it to have several great properties, expecting them to work effectively in delivering drugs in vivo. However, the designed carrier may not function as intended once in the body.

Table 1 Summary of FDA-Approved Nanocarrier Drugs to Date (Grouped by Nanocarrier Type, Including Discontinued Nanocarrier Drugs)a

When developing nanocarriers, formulation design and subsequent pharmacological experiments are usually performed by different teams, which poses a major problem as front-end pharmaceutical researchers, although having theoretical knowledge and insights into carrier design, often lack a thorough understanding of the carrier’s in vivo fate and mechanisms compared to pharmacologists. The process of bringing a nanocarrier to market requires a substantial investment in terms of money, human resources, or material resources. If the drug has poor clinical effects or safety issues, it can result in significant losses owing to the need for rework or abandonment. Therefore, in the early stage of nanocarrier design, we should consider the clinical impact, which requires researchers to have sufficient knowledge about the in vivo fate of nanocarriers.

Therefore, pharmacy workers and others involved in nanocarrier research should first study the fate of the carriers in vivo, increase the investment in the study of the fate of the blank carriers in vivo, and firmly grasp the fate of the carrier in vivo. By doing so, the success rate of pharmacological experiments and clinical translation can also be improved, benefiting patients by providing drugs for their conditions promptly.

The above was also our intention while conducting this study, which aimed to provide an overview of the in vivo fate of delivery carriers with targeting capabilities that have been reported. We aimed to compare the subject matter of this study with other studies on similar topics about drug delivery carriers (Table 2).

Table 2 Theme Comparison of This Review Article with Other Review Articles on Similar Topics (with Examples of Typical Articles)

In conclusion, this article aimed to summarize the in vivo fate mechanisms of 13 major classes of targeted drug delivery carriers that have undergone some research progress. Specifically, this compilation elaborates on their major tissue distribution upon entry into the body, blood circulation time, interaction mechanisms with cell membranes, intracellular transport methods, escape capabilities, degradation and excretion pathways, and potential toxicity. Despite the existence of undefined aspects in the in vivo fate mechanism of the targeted delivery drug carriers summarized in this article, this study can provide valuable references for researchers in selecting targeted drug delivery carriers and gaining a preliminary understanding of their in vivo distribution.

Types of Targeted Drug Delivery Carriers

Presently, various carriers are used for targeted drug delivery. We broadly categorized the delivery carriers with targeting capabilities reported into 13 major categories (Figure 1). We organized their common carriers (We listed the specific carrier categories commonly used, and analyzed these commonly used carriers. The commonly used carrier categories was not listed because they are not generalized designations, belonging to a specific class of carriers, and are subsequently used directly in the analysis), meaning, property, role, the types of drugs that can be transported and their main fields of application, and created a table (Table 3).

Figure 1 Schematic diagram of targeted drug delivery carriers.

Table 3 Summary of a Brief Description of the 13 Major Classes of Targeted Drug Delivery Carriers

Biodistribution

Small molecule drugs are often quickly eliminated by the kidneys upon entering the bloodstream, potentially reducing their efficacy. However, when drugs are loaded into carriers, their circulation half-life is prolonged, leading to enhanced accumulation at the targeted site and promoting therapeutic efficacy. Various targeted drug delivery carriers exhibit unique biological distributions, and the following section discusses three main aspects of the distribution for each carrier.

Targeting

Targeted drug delivery is a promising research approach for treating diseases such as cancer, aiming to deliver therapeutic drugs precisely to the affected site and minimize damage to healthy tissue. This approach improves drug efficacy while reducing side effects.3

Targeted drug delivery can be achieved through three main strategies: 1) passive targeting, primarily based on the carrier’s size, and Enhanced Permeability and Retention (EPR) effect. The EPR effect refers to high permeability and prolonged retention of substances <200 nm in solid tumors. The lack of vascular support tissue, poor structural integrity, and large inter-endothelial cell gaps in tumors allow carriers <200 nm to easily penetrate the vascular wall and enter tumor gaps. Furthermore, tumors lack lymphatic vessels, preventing the carried drugs from being drained and enabling long-term accumulation.59–61 2) Active targeting mode: addition of targeting ligands. Targeting ligands are substances with a high affinity for the target site, including folate, transferrin, peptides, antibodies, aptamers, and oligosaccharides. These ligands bind to specific receptors overexpressed in the lesion tissue, facilitating targeted drug delivery.61,62 3) Physicochemical targeting mode: using physical and chemical methods to enable targeted formulations to exert their therapeutic effects in specific locations. The carriers prepared via this method are stimulus-sensitive carriers, which undergo rapid changes when exposed to external stimuli (such as light, magnetic fields, high temperature, ultrasound) or special target site environments (such as enzyme overexpression, low pH), releasing the drugs they carry and achieving accurate drug release at the target site.63,64

Passive Targeting

Carriers based on passive targeting patterns are liposomes,65 lipid nanoparticles (LNPs),66 albumin carriers,67 follicular dendritic cell (FDC),59 dendrimers,24 micelles/ polymeric micelles (PMs),68,69 gold nanoparticles (AuNPs),37 nanogels,70 microbubbles,71 angelica sinensis polysaccharide (ASP),43 extracellular vesicles (EVs),72 adeno-associated viral (AAV) vectors.73 Carriers that target specific sites without adding ligands belong to the passive targeting strategy. The targeting mechanism of passive targeting carriers is as follows.

Liposomes, Dendrimers, and LNPs

Carriers with a diameter <200 nm, such as liposomes and dendrimers, are easily passively targeted to tumors because the endothelial walls of healthy tissues are tightly connected, preventing the carriers from entering. In contrast, tumors have larger inter-endothelial gaps and more porous capillaries, resulting in higher permeability. The amount of carriers taken up by tumors can be >100 times higher than that of healthy tissues.24,59,74,75

LNPs can bind with apolipoprotein E (ApoE) in plasma to form ApoE-bound lipoproteins, which are mainly cleared by the liver, allowing LNPs to reach the liver preferentially for liver targeting. This method is commonly used to deliver RNA to the liver using cationic or ionizable LNPs. The presence of ionizable lipids and intentional short acyl chain PEG lipids enhance the ability of LNPs to deliver RNA compared to liposomes. Liver targeting is insufficient for treatment, as specific cell targeting is often necessary. Therefore, Kim et al modified the overall size of LNPs by adjusting the polyethylene glycol (PEG) content, specifically targeting hepatocytes.76 Lipid nanoparticles (LNPs) can be modified to selectively target specific cell types. They offer the benefits of delivering mRNA with high transfection efficiency, high expression levels, and good stability, making them crucial carriers for mRNA vaccine development.77

Albumin Carriers and FDC

Albumin carriers can specifically bind to cell surface albumin receptor (gp60), which binds to intracellular caveolin-1, leading to membrane invagination and the formation of caveolae. Caveolae can transport albumin carriers to tumors, making carriers bind to secreted protein acidic and rich in cysteine protein overexpressed in tumors, thereby targeting the albumin carriers to the tumor.78 The tumor-targeting effect of ferritin is ten times greater than that of the EPR effect. Ferritin does not require surface modification as it naturally targets tumors through its specific affinity for transferrin receptor 1 (TfR1), which is overexpressed in malignant tumors. Ferritin is the optimal size of 12 nm for inducing the EPR effect caused by disrupting the blood and lymphatic systems in tumors.79

AuNPs, Nanogels, and ASP

AuNPs, after being injected into the body, can accumulate in inflammatory and tumor sites through the enhanced permeability and EPR effect to achieve passive targeting.37

Nanogels also accumulate at the site of lesions through the enhanced permeability and EPR effect to achieve passive targeting. However, the targeting capabilities of these nanogels are low; therefore, current nanogels are mostly designed as active targeting agents by adding ligands and other methods.70

ASP has a high affinity for the asialoglycoprotein receptor that is highly expressed in the liver, allowing for liver targeting.43

EVs, Yeast, and AAV Vectors

EVs can cross physiological barriers and target tissues through their surface protein markers.80 However, some problems are encountered in the targeting design of EVs because of the complex composition and naturally highly heterogeneous of EVs, which influence the therapeutic effect.81

The mechanism of specific targeting of phagocytes by yeast carriers is unique. The β-glucan in the yeast cell wall can specifically bind to pattern recognition receptors on phagocytes, thereby targeting phagocytic cells.55

AAV vectors, such as AAV8, target the liver, and AAV1 and AAV5 target cells in the central nervous system.73

Active Targeting

Carriers based on active targeting patterns are biomimetic nanoparticles and carriers modified with folate, transferrin protein, peptides, antibodies, aptamers, hyaluronic acid (HA), PEG, and oligosaccharides.49,62,82

Folate, transferrin protein, peptides, antibodies, aptamers, HA, and oligosaccharides can bind with specific receptors overexpressed in lesions, allowing carriers to target lesion sites. For example, folate receptor (FR) and TfR in tumors are responsible for the uptake of folate and iron, respectively. Tumors need a large amount of folic acid and iron during rapid proliferation. Folate and transferrin can bind to FR and TfR expressed at high levels in cancer cells with high affinity, mediating carriers modified with folate or transferrin to target tumor tissues, respectively.83,84 HA can bind to CD44 receptors that are significantly expressed in cancer cells, targeting carriers modified with HA to cancer cells.85

The emerging targeting strategy is to load drugs for precisely targeted delivery by using vesicles from disease cells or some cell membranes that contain receptors on their surface that bind specifically to cells at the site of the disease as carriers. For example, Yon et al86 developed a bio-compatible tumor cell-derived extracellular vesicle biomimetic porous silicon nanoparticle as a drug carrier for targeted cancer therapy (Figure 2). This carrier exhibits enhanced tumor accumulation and penetration into the deep tumor. The developmental idea of using biomimetic extracellular vesicle nanoparticles derived from tumors as drug carriers for targeted chemotherapy in cancer is a proof-of-concept and provides new design ideas for researchers developing targeted delivery drug carriers.

Figure 2 (a and b) Schematic diagram of E-PSiNPs as drug carriers for targeted cancer chemotherapy. (c) Transmission electron microscope images of PSiNPs and E-PSiNPs. (d) Hydrodynamic diameter of E-PSiNPs incubating in phosphate-buffered saline with or without 10% fetal bovine serum for different intervals. (e) DOX content in tumor tissues and major organs of H22 tumor-bearing mice 24 h after intravenous injection. (f) Penetration of DOX@PSiNPs or DOX@E-PSiNPs into tumor parenchyma. Reprinted from Yong T, Zhang X, Bie N, et al. Tumor exosome-based nanoparticles are efficient drug carriers for chemotherapy. Nat Commun. 2019;10(1):3838. Creative Commons.86

Yan et al87 prepared a penetrating macrophage-based nanoformulation (MZ@PNM)-encapsulating hydrogel (MZ@PNM@GCP) that responded to the periodontitis microenvironment. The loaded nanoparticles (MZ@PNM) could specifically target porphyromonas gingivalis, a key mediator of periodontitis, through their Toll-like receptor 2/1 mimicking membrane on macrophages. Subsequently, the macrophage membrane fused with the bacterial outer membrane, and the cationic nanoparticles within MZ@PNM disrupted the bacterial structure and released metronidazole in the bacterial cytoplasm (Figure 3).

Figure 3 (a) Schematic diagram of the action of MZ@PNM in targeting and killing P.g. (b–c) Zeta potential and diameter characterization of MZ@PNM and MZ@PNP. ***p < 0.001. (d) Representative TEM image of the nanomacrophages targeting P.g. (e–f) Representative fluorescence image and TEM images of the destruction of P.g. by MZ@PNM over time. Reprinted from Yan N, Xu J, Liu G, et al. Penetrating Macrophage-Based Nanoformulation for Periodontitis Treatment. ACS Nano. 2022;16(11):18253–18265. Copyright © 2022, American Chemical Society.87 P.g., Porphyromonas gingivalis; TEM, Transmission electron microscope.

Physicochemical Targeting

When the physicochemical target carriers are exposed to external stimulation or are in a target site environment, they will rapidly change and release their loaded drug to achieve the effect of precise drug release at the target site. For example, thermo-sensitive liposomes, photo-sensitive liposomes, ultrasound-guided carriers, and pH-sensitive carriers are all based on this design.82 Thermo-sensitive liposomes are sensitive to temperature, photo-sensitive liposomes are sensitive to light, and ultrasound-guided carriers are guided by ultrasound, all of which are precisely delivered to target sites under external stimulation. The mechanism of pH-sensitive carrier release is that in different pH environments inside the body, pH-sensitive carriers are sensitive to the pH of a specific environment and undergo structural or other changes, thereby releasing the loaded drugs in that location, achieving precise drug release. Generally, there are two types of pH-sensitive carriers. One type is polymers containing ionizable functional groups, which protonate owing to pH changes, resulting in changes in hydrophilic–hydrophobic balance and changes in structure or solubility. The other type has acid–labile bonds; therefore, they are cleaved in acidic environments, leading to changes in carrier structure and drug release.88 For example, the pH of plasma, the tumor microenvironment, and the pH of lysosomes and nuclei are neutral (6.5–7.0) and acidic (4.5–5.5), respectively. Therefore, carriers sensitive to an environment with a pH of 6.5–7.0 can release drugs precisely in tumor environments, achieving tumor targeting.89,90

Moreover, carriers with dual sensitivity can be combined to achieve dual targeting control, such as thermo-sensitive magnetic liposomes, which are thermally sensitive and magnetically guided, making them more suitable for the special properties of the lesion location.82

In addition to carriers with inherent targeting properties, other carriers mostly need to be modified on the surface to have targeting properties to cells and tissues. Researchers have also discovered some materials that are very suitable for developing targeted carriers, such as selenium nanoparticles (SeNPs),91 graphene and its composite materials,92 layered double hydroxide (LDHs),93 quantum dots (QDs),94 chitosan,95 adenovirus vectors,96 and lentiviral vectors.97

Different targeted drug delivery carriers have different targeting mechanisms, and selecting targeting carriers can be based on the site and characteristics of the disease to be treated. For example, we can choose a carrier that can specifically bind to the lesion or passively target the lesion, or we can modify a carrier with better performance to make it actively target the lesion. Furthermore, a responsive targeting vector can be designed to target some focal sites with special environments. If the microenvironment of a focal site has a low pH, a targeting vector sensitive to low pH can be designed so that it is released at that site but not in a normal pH environment. Precisely targeted carriers for release can also be achieved by designing carriers externally controlled or stimulated by delivering the carrier through control or stimulation such as light, heat, temperature, and magnetic field.

Major Tissue Distribution

Generally, carriers with a diameter <5 nm are easily cleared from the circulation, while carriers >50 nm accumulate non-specifically in the liver, which has fenestrations with a length of 50–100 nm. Carriers with a diameter >200 nm accumulate in the spleen, which has fenestrations with a length of 200–500 nm.2,98,99 However, the specific distribution of particles with diameters ranging from 20–150 nm is also influenced by their charge and shape. For example, among particles of this size, those shaped like cylinders or discs are more likely to distribute to the liver and spleen.98 Below is a summary of the major tissue distribution of various carriers.

Liposomes, LNPs, and Graphene Materials

Conventional liposomes and LNPs, which are carriers composed of lipids, are recognized and taken up by the reticuloendothelial system (RES) after intravenous injection, with most accumulating in the liver and spleen (RES organs), and after oral administration, they are hydrolyzed in the gastrointestinal tract, with only a few remaining carriers able to circulate to the target site.11,14,63 After LNPs are administered through the intramuscular route, they first accumulate in the muscle tissues, then enter the systemic circulation through the lymphatic system and the blood circulatory system, part of which will be taken up by the RES organs such as the liver, and the remaining will reach the corresponding target site to deliver mRNA according to the design.100

Graphene and graphene oxide are mainly distributed in the liver after entering the body and are taken up by the RES.101

EVs

EVs entering the body are primarily accumulated in the liver, followed by the spleen, gastrointestinal tract, and lungs, although the actual distribution of EVs from different cells and in different amounts may vary.102 For example, ginger-derived extracellular vesicles remain in the gastrointestinal tract for a long time after oral administration, while grape fruit-derived extracellular vesicles mainly accumulate in the lungs and brain after nasal administration.103–105 Additionally, exosomes accumulate most in the liver, followed by the spleen, gastrointestinal tract, and lungs, although excessive accumulation of exosomes in the liver is relatively low owing to the phagocytic system’s uptake threshold.106

PEG-modified carriers affect carrier distribution in tissues, reducing steric hindrance, forming a barrier to prevent the modulatory effect of serum proteins, reducing phagocytosis by the RES, and protecting the carrier from uptake by the liver.64

Blood Circulation Time

When the carriers enter the bloodstream, they will adsorb plasma proteins, such as serum albumin, lipoproteins, complement components, and immunoglobulins, and form a protein corona on its surface. This structure makes the carrier more prone to attaching to specific receptors on the surface of phagocytes for phagocytosis, thereby shortening its circulation time in the bloodstream and affecting its stability and fate in the body. Additionally, the circulation time of the carrier in the bloodstream is also related to its shape. For example, filamentous polymer micelles align with the blood flow owing to their shape, resulting in longer circulation times than spherical polymer micelles.98,107 Therefore, different targeted drug delivery carriers have different circulatory times in vivo. The following are discussions on the circulatory time of various carriers in vivo.

Liposomes, LNPs, Dendrimers, and Micelles/PMs

Conventional liposomes are easily eliminated by the RES owing to their easy uptake, resulting in a short circulatory time.63 Newly developed long-circulating liposomes, which combine liposomes with some chemically and biologically inert synthetic polymers, can prolong the circulatory time of drugs in vivo.2

NPs with a diameter of 10–100 nm are not easily engulfed by the RES and generally have longer circulatory times, while larger NPs are easily engulfed by the RES and have shorter circulatory times.2 LNPs with a diameter <200 nm are not easily taken up by the RES and are cleared by the RES, resulting in a longer circulatory time.99

Conventional dendrimers have a short circulatory time owing to their small volume of 10 nm, which makes them easily cleared in vivo.24 Micelles and PMs have a hydrophilic outer shell and are 10–100 nm in diameter, making them not easily taken up and cleared by the RES, resulting in a longer blood circulation time.68,108,109

Albumin Carriers

Endogenous albumin circulates for approximately 19 days because when it enters the endothelial cells, the FcRn receptors in the endothelial cells expel the albumin from the cells via exocytosis, and the albumin returns to the bloodstream through the lymphatic system, resulting in prolonged circulation. However, if albumin is made into albumin nanoparticles or modified into drugs, the circulation time will be reduced to an extent.67

AuNPs, Graphene Materials, QDs, Nanogels, and Microbubbles

AuNPs have a short plasma half-life, which is mainly influenced by two substances in the body that affect the carrier’s half-life, namely opsonins and dysopsonins. Opsonins (such as complement and immunoglobulin supplements) can promote the rapid engulfment of liposomes via the RES, while dysopsonins (such as serum albumin and lipoprotein) can improve the circulation half-life of liposomes by preventing engulfment.74 Opsonins are easily adsorbed onto the surface of AuNPs, promoting the clearance of AuNPs by phagocytes, resulting in a short half-life for AuNPs.89 Graphene and graphene oxide are easily cleared from the blood, resulting in a short circulatory time.110 QDs’ coating materials have certain immunogenicity, which can easily cause an immune response in the body, leading to the clearance and ineffectiveness of QDs, resulting in a short half-life. However, if low-toxicity materials such as carbon and silicon are used for preparation, their circulation time can be prolonged.111

Nanogels with a size of 20–200 nm are not easily engulfed by the RES and have a longer blood circulation time.33 Using ultrasound-mediated microbubble carriers can significantly prolong their circulation time.71

Biomimetic Nanoparticles, EVs, and Viral Vectors

Red blood cell membrane coating and platelet membrane encapsulation can reduce the uptake of nanoparticles via the RES and extend their circulation in the bloodstream.44,112 EVs have a long circulation time because they are endogenous and are not easily cleared by the immune system.72

Owing to the expression of non-deleted viral genes and ensuing immune responses to the expressed viral proteins, the circulation time of Ad vectors in the bloodstream is relatively short.73 Among them, chimpanzee-derived Ad vectors have the longest circulation time because the level of neutralizing antibodies against them in the body is the lowest.56

Moreover, modifying the carriers can also change the circulation time in the bloodstream. For example, coating the carrier surface with PEG to form a PEG-coating can provide a protective barrier to prevent carrier lipolysis when entering the gastrointestinal tract. The ethylene glycol units of PEG coating can form a hydrating layer by tightly binding with water molecules. This hydration layer can prevent protein adsorption and recognition by RES, thereby extending the half-life of drugs and increasing their circulation time.11,85,113,114 Modifying the carrier with dextran can also protect it and prolong its circulation in the bloodstream. This benefits the drug accumulation around the target tissue or cells to enhance its effectiveness.115

In addition, nanomotors116 and cell carriers117 can prolong the circulation of drugs in the blood. Since the data obtained from different studies are different, and the circulation time of some carriers is not recorded, it is inaccurate to directly state the long or short circulation time of the carriers, so we have compiled a table on the expected pharmacokinetic parameters and biodistribution of the carriers (Table 4) for the readers’ reference.

Table 4 Summary of General PK and Major Tissue Distribution of Different Types of Carriers

Fate of Targeted Drug Delivery Carriers at the Site of Lesions

In the previous section, we discussed how targeted drug delivery carriers can distribute to different tissues and organs after entering the human body. Thereafter, carriers face the problem of releasing drugs. Different carriers have different mechanisms for drug release. Some carriers may release drugs at the site of lesions without entering cells, while others may only interact with cell membranes and release drugs into the cytoplasm but not enter the cells. Some carriers can enter cells through certain pathways and be transported at the subcellular level through certain pathways. Some cannot escape the fate of degradation, while others can escape. The following is a discussion of the fate of each carrier at the site of lesions. The carriers discussed in this section are carriers in their complete form that can reach the site of lesions.

Fate of Carriers That Interact with Cell Membranes

There are two main types of interactions between carriers and cell membranes (Figure 4): 1) endocytosis, which refers to transporting extracellular substances into cells through deformation movements of the plasma membrane. This process requires entry into the cell, and carriers that enter cells through endocytic pathways will reach lysosomes or endosomes for degradation. Some carriers will escape after reaching endosomes or lysosomes to avoid degradation; 2) membrane fusion, which refers to the ability of the carrier’s membrane to fuse with the cell membrane, allowing the loaded drugs to be directly released into the cytoplasm without entering the cell. Drugs delivered through this process will not enter endosomes or lysosomes and do not need to undergo escape.143,144

Figure 4 Schematic diagram of mechanisms of endocytosis and membrane fusion. (a) Adsorptive endocytosis. (b) Clathrin-mediated endocytosis. (c) Receptor-mediated endocytosis. (d) Caveolae-mediated endocytosis. (e) Phagocytosis. (f) Membrane fusion.

Carriers for Cellular Entry by Endocytosis

There are several pathways for endocytosis of targeted drug delivery carriers: (1) adsorptive endocytosis; (2) clathrin-mediated endocytosis; (3) receptor-mediated endocytosis; (4) caveolae-mediated endocytosis; (5) phagocytosis.2,143,145 Not all endocytosis pathways were covered. Clathrin-mediated-mediated endocytosis and pinocytosis were not covered. Listed here are only the explicitly documented endocytosis pathways for all targeted delivery carriers summarized in this study.

In general, carriers that enter cells by endocytosis first enter the lysosomes or endosomes. The exception to this is caveolae-mediated endocytosis, in which carriers entering the cell are transported via a non-lysosomal pathway within the cell for reasons explained in detail in the section on caveolae-mediated endocytosis. Lysosomes and endosomes contain more than 60 acidic hydrolases, which are inactive enzymes that become active when drugs enter the lysosomes or endosomes and can degrade the incoming substance. This process occurs at a pH of about 5. The pH in lysosomes and endosomes is about 4.5–5.5, with pH changes occurring before and after degradation. Therefore, if the carrier is required to transport drugs to the nucleus or the degradation of carriers in lysosomes or endosomes is not desired, carriers with “endosomal/lysosomal escape” properties can be selected, or carriers can be designed to have “endosomal/lysosomal escape” capability.75 “Endosomal/lysosomal escape” may not be the only way to prevent carriers from being degraded by endosomes or lysosomes; however, it is currently the most widely used method. There are generally two mechanisms by which nanoparticles can escape the endosome or lysosome upon internalization (Figure 5): (1) Expansion-induced variation, where the nanoparticle carrier dissociates within the endosome/lysosome, causing mechanical strain that leads to rupture of the organelle, thereby releasing the carrier and its cargo into the cytoplasm, followed by cytoplasmic transport of the nanoparticles; (2) Proton sponge effect, where the pH within the endosome/lysosome decreases, allowing cationic polymers within it to capture several protons. To maintain charge and concentration balance, external Cl− and water molecules flow into the organelle, resulting in increased permeability and eventual rupture of the organelle, thereby releasing the cargo or carrier into the cytoplasm and achieving escape.146 The following is a classification of various carriers’ cellular uptake, subcellular level transportation, and drug release mechanisms, mainly based on endocytosis.

Figure 5 Schematic diagram of the action of the endosomal escape mechanism. (a) Carboxylic acid, phosphoric acid, amino, and other functional groups of pH-sensitive nanogel undergo dissociation in the acidic environment of the endosome. At this point, external water enters the nanogel, causing it to swell, rupture the endosome, and release the enclosed drugs into the surrounding nanogel to achieve endosomal escape. (b) The pH of the endosome decreases between the early and late stages, and cationic polymers can buffer the pH drop of endosomes and trap H+ after entering endosomes. At this time, to maintain charge balance and concentration balance, Cl− and water molecules will flow into the endosome in large quantities, causing the endosome to burst, and the drugs and carriers in them are released into the cytoplasm to achieve endosomal escape.

Cellular Entry Mechanism, Subcellular Level Transport Pathway, and Drug Release Mechanism of Carriers Entering Cells via Adsorptive Endocytosis

Adsorptive endocytosis is a process of cellular uptake of external substances, which are adsorbed onto the cell membrane by interacting with the charge, hydrophobicity or other physicochemical properties of the cell membrane. Subsequently, the cell membrane depresses and wraps around these substances, forming endocytosis vesicles that transport them inside the cell for further processing. It is similar to receptor-mediated endocytosis, but differs in that adsorptive endocytosis is not dependent on specific cell surface receptors.147,148 Currently, carriers that enter cells through adsorptive endocytosis are cationic liposomes147 and chitosan.149 The mechanisms are as follows.

Cationic liposomes with ionizable lipids can electrostatically bind to the negatively charged cell membrane, enter the cell through adsorptive endocytosis, and become a vesicle engulfed by the cell membrane. Thereafter, it detaches from the cell membrane and enters the cytoplasm. Subsequently, it is transported through the endosome-lysosome pathway in the cell. In the acidic environment of the endosome, cationic liposomes are protonated, allowing them to interact with ionizable lipids in the bilayer of the endosome, forming a non-bilayer hexagonal structure that disrupts the bilayer. This releases the drug in the lipid into the cytoplasm.74,147 Additionally, combining cationic liposomes with primary or secondary amines with positive charges can help them escape from lysosomes, thereby protecting drug molecules.74

Chitosan with positive charges can adhere to the negatively charged cell membrane surface via electrostatic interaction, enter cells through adsorptive endocytosis, and reach the endosomes. Owing to its positive charge, it can undergo endosomal escape, allowing the release of loaded drugs into the cytoplasm. However, the endosomal escape ability of chitosan is inefficient; therefore, researchers often modify it to enhance this capability.149

Cellular Entry Mechanism, Subcellular Level Transport Pathway, and Drug Release Mechanism of Carriers Entering Cells via Clathrin-Mediated Endocytosis

The mechanism of clathrin-mediated endocytosis is that when some receptors on the cell membrane surface are bound, the clathrin coat protein in the cytoplasm aggregates on the membrane, and the adaptor protein makes the membrane bend, enclosing the carrier into a clathrin-coated pit. Thereafter, it invaginates and enlarges and finally breaks off, allowing the carrier to be transported into the cell.144 The carriers that enter cells through clathrin-mediated endocytosis include graphene/graphene oxide,101 LDHs,93 and AAV vectors.56,73 The mechanism is as follows:

Positively charged LDHs penetrate the negatively charged cell membrane through clathrin-mediated endocytosis.93 Thereafter, LDHs enter the endosome, where they partially dissolve and release metal ions. Several water molecules in the cytoplasm enter the endosome, causing it to burst. The drug, with some LDHs, is released into the cytoplasm.34 When AAV vectors reach the cell surface, they first bind to sugars (such as sialic acid, lactose, or heparan sulfate) and AAV receptors on the cell surface and enter the cell through clathrin-mediated endocytosis. Thereafter, they reach the endosome and are transported through the late endosome and lysosome compartments. During this process, AAV vectors undergo endosome/lysosome escape. Finally, the genetic material in the carrier is transported to the nucleus and exerts its effect.56,73

Cellular Entry Mechanism, Subcellular Level Transport Pathway, and Drug Release Mechanism of Carriers Entering Cells via Receptor-Mediated Endocytosis

The mechanism of receptor-mediated endocytosis is that when the carrier reaches the cell surface, it specifically binds to receptors on the surface to form a complex. Thereafter, the membrane invaginates to form a coated pit, which detaches from the membrane to form a coated vesicle, taking up the extracellular carrier.145 The carriers that enter cells through receptor-mediated endocytosis are FDC78 and nanogels containing pullulan.33 The mechanism is as follows.

FDC specifically binds to TfR overexpressed on the surface of tumors and enters cells through TfR-mediated endocytosis. Subsequently, FDC is transported through the endosome-lysosome pathway, where FDC undergoes reversible decomposition and releases the drug in the acidic environment of the endosome and lysosome.78,79

Nanogels containing pullulan can strongly bind to salivary glycoprotein receptors and enter cells through receptor-mediated endocytosis. The mechanism of drug release from nanogels is a phenomenon called the “shape-memory effect.” In the acidic environment of the endosome, the nanogel swells, causing the endosome to rupture. Thereafter, water enters the nanogel, releasing the drug into the cytoplasm. When in the neutral cytoplasm, the nanogel shrinks to its original size, retaining unreleased drugs within the nanogel, waiting for the next release cycle. The process of drug release consists of two mechanisms: “swelling” and “permeation”.33,150

Cellular Entry Mechanism, Subcellular Level Transport Pathway, and Drug Release Mechanism of Carriers Entering Cells via Caveolae-Mediated Endocytosis

The mechanism of caveolae-mediated endocytosis is that the carrier reaches the cell membrane surface, covered by caveolin, to form a vesicle and enter the cell. This process bypasses lysosomes, avoiding drug degradation.151 Caveolae-mediated endocytosis typically allows particles sized 20–100 nm to enter the cell, with a maximum particle size limit of 200 nm. Particles larger than 200 nm may enter the cell through alternative pathways.152,153 Most endocytic processes are energy-dependent. Carriers that enter the cell through these endocytic processes are first transported to lysosomes or endosomes for degradation, destroying the carriers before they can deliver the loaded drugs to the cytoplasm or nucleus. Unless carriers can escape lysosomes/endosomes, it would be better if carriers could directly bypass lysosomes or endosomes. Caveolae-mediated endocytosis is an excellent way to achieve this. Carriers entering the cell via this pathway can bypass the lysosome-endosome pathway of cytoplasmic delivery through membrane insertion and diffusion and reach the cytoplasm directly. It can avoid degradation of carriers and loaded drugs in lysosomes or endosomes and improving drug retention and efficacy. However, the only carriers that enter the cell via caveolae-mediated endocytosis are graphene QDs (GQDs) and carbon nanotubes.154,155 GQDs are distributed in the endoplasmic reticulum and nucleus after entering the cell,155 and how drug release occurs is unclear. The fate of carbon nanotubes after reaching the cytoplasm is still unknown. This field has great development potential, and to avoid carriers degraded by lysosomes or endosomes, carriers can be designed to enter the cell by caveolae-mediated endocytosis.

Zhang et al156 have successfully designed nanoparticles that enter cells through this pathway. They developed a core-shell nanocarrier coated by cationic albumin (CNC), which can simultaneously deliver miRNA-34a and docetaxel into breast cancer cells for synergistic therapeutic effects. CNCs can effectively protect miRNA-34a from degradation by RNase and serum, and they can also enter the cytoplasm via caveolae-mediated endocytosis, avoiding degradation by endosomes or lysosomes and improving cargo utilization efficiency (Figure 6a–d). While carriers that enter the cell by other endocytosis first undergo lysosomal or endosomal transport pathways at the subcellular level (Figure 6e).157 Carriers without escape capability are easily degraded in lysosomes or endosomes, and carriers with escape capability have different ways or degrees of lysosomal or endosomal escape according to their respective escape mechanisms (Figure 6f).158

Figure 6 (a) Schematic illustration of CNCs co-delivering DTX and miRNA-34a. (b) Confocal laser scanning microscopy images of intracellular fluorescence of CNCs in 4T1 cells pretreated with various inhibitors. (c) Colocalization of C6CNCs with caveolae after incubation in the absence or presence of nystatin. (d) Cytosolic location of the CNCs in 4T1 cells observed using TEM after incubation. Reprinted from Zhang L, Yang X, Lv Y, et al. Cytosolic co-delivery of miRNA-34a and docetaxel with core-shell nanocarriers via caveolae-mediated pathway for the treatment of metastatic breast cancer. Sci Rep. 2017;7:46186. Creative Commons.156 (e) Confocal imaging of 200 nM FITC (green)-conjugated AfFtnAA uptake by RAW264.7 cells after 24 h of incubation. Reprinted from Ravishankar S, Nedumaran AM, Gautam A, Ng KW, Czarny B, Lim S. Protein nanoparticle cellular fate and responses in murine macrophages. NPG Asia Materials. 2023;15(1):1. Creative Commons.157 . https://creativecommons.org/licenses/by/4.0/. (f) TEM images of mouse embryonic fibroblasts incubated with listeriolysin O H311A-NPs. Reprinted from Plaza-Ga I, Manzaneda-González V, Kisovec M, et al pH-triggered endosomal escape of pore-forming Listeriolysin O toxin-coated gold nanoparticles. J Nanobiotechnology. 2019;17(1):108. Creative Commons.158 TEM, Transmission electron microscope; CNC, cationic albumin; DTX, Docetaxel.

Cellular Entry Mechanism, Subcellular Level Transport Pathway, and Drug Release Mechanism of Carriers Entering Cells via Clathrin-Mediated Endocytosis and Phagocytosis

In addition to the above carriers, some carriers have multiple mechanisms, generally two.

Phagocytosis refers to the cell engulfing the carrier.159 LNPs are absorbed by cells through clathrin-mediated endocytosis and phagocytosis. Thereafter, they are transported through the endosome-lysosome pathway in cells.11 During the endocytosis, the positive charges on the cationic LNPs can be neutralized and disrupted by the negative lipids of the cells, leading to the release of the encapsulated drugs.160

Cellular Entry Mechanism, Subcellular Level Transport Pathway, and Drug Release Mechanism of Other Carriers Entering Cells via Endocytosis

In addition to the carriers discussed above, some carriers only enter the cell through endocytosis; however, the specific endocytic pathway is unknown.

Micelles enter the cell through endocytosis and reach the endosomes, where the micelles may degrade into monomers, may also degrade in advanced endosomes, or may escape from endosomes.151,161 EVs112 and yeast55 enter cells through endocytosis. Ad vectors can enter cells through endocytosis, reach the endosome, and then the viral shell is decomposed. Thereafter, V and VI proteins help the viral genetic material escape from the endosome and enter the cell nucleus through the nuclear envelope.56

In addition, nanocarriers containing fusogenic, pH-sensitive, or cationic lipids can escape from the endosome pathway to the cytoplasm.151

Carriers for Interaction with Cell Membranes via Membrane Fusion

The representative vectors that act with the cell membrane through membrane fusion and release the drug into the cytoplasm are lentiviral vectors.56 The glycoproteins on the envelope of lentiviral vectors bind to corresponding receptors on the cell membrane, causing the viral membrane to fuse with the cell membrane and directly release the genetic material inside into the cytoplasm.56,162 The spikes on the surface of the viral vector can also help it bind strongly to the cell membrane.163 The invasion of genetic information of lentiviral vectors mainly includes two processes: reverse transcription and integration. After the viral shell is removed, viral nucleic acids and protein complexes that enter the cytoplasm are key components for subsequent genetic information to be integrated into chromosomal DNA, often called reverse transcription complex (RTC). Viral RNA is first reverse-transcribed into double-stranded viral DNA in RTC and transported to chromosomal DNA, where it is integrated.162

Carriers for Interaction with Cells via Endocytosis and Membrane Fusion

The above describes the mechanisms by which carriers interact with cells through endocytosis and membrane fusion. These carriers belong to either of these mechanisms; however, some carriers may interact with the cell membrane, such as microbubbles, via both mechanisms. There are two main mechanisms by which microbubbles enter cells: 1) they are engulfed by the cell membrane; 2) they enter the cell through membrane fusion, where the phospholipid component of the microvesicle merges with the double-layered phospholipid of the cell membrane, directly delivering the loaded drug into the cytoplasm.71

Others

The cellular uptake of AuNPs and SeNPs is poor.89,91 Micro/nanomotors can quickly internalize into cells and are not prone to be captured and degraded by lysosomes.164 Targeting ligands influence the pathway of carrier endocytosis by binding specifically to receptors on the cell membrane. When a carrier has a cognate receptor on the target cell or is modified with a ligand that binds to a receptor on the target cell membrane, it enters the cell via receptor-mediated endocytosis. For instance, if a carrier is modified with albumin, it will bind to the albumin receptor on the target cell and enter via caveolae-mediated endocytosis. Similarly, ferritin-modified carriers bind to the TfR1 receptor on tumor cells, allowing entry through receptor-mediated endocytosis. The endocytosis pathway can vary even with the same targeting ligand if different cells are targeted. For example, entry via lipid raft-mediated endocytosis requires the presence of lipid rafts on the cell membrane, with the niche being a specific type of lipid raft.78,79

Fate of Carriers That Do Not Interact with Cell Membranes

Not all carriers can transport drugs into cells. Some carriers may change when they reach the target site and release drugs outside cells, such as neutral liposomes and physicochemical targeting carriers. Neutral liposomes have no significant interaction with cells; therefore, their drug release mainly occurs in the extracellular space. When physicochemical targeting carriers reach the target site, they undergo a certain stimulus from the external environment or are sensitive to the specific environment, and the carrier will change, allowing it to release the loaded drugs. For example, around tumors, rapid anaerobic glycolysis produces excessive protons and carbon dioxide. The tumor lacks a functional lymphatic drainage system, resulting in insufficient oxygen supply and excessive lactate secretion, leading to extracellular acidification (the Warburg effect). Therefore, the extracellular pH around tumors is generally between 6.8 and 7.0, and in some cases, even as low as 5.7. For pH-sensitive carriers that are sensitive to such pH conditions, when they reach the tumor site, their lipids, organic functional groups, or inorganic compounds can easily change shape and release the loaded drugs. Poly (methacrylic acid), N-isopropyl acrylamide, poly (diethylaminoethyl methacrylate), poly (acrylamide), and poly (acrylic acid) are key components of pH-sensitive carriers that respond to pH changes, namely “pH-responsive membrane-deshielding type polymers.” Under acidic pH conditions, pH-responsive membrane-deshielding type polymers with carboxylic groups are protonated to eliminate electrostatic forces. The hydrophobicity of the polymer increases, and the polymer transforms from a hydrophilic state to a hydrophobic state, enhancing the interaction between the carrier and cell membranes and promoting drug release.63

Clarifying how the carrier interac