Nanocarrier-Based Drug Delivery Systems Targeting Kidney Diseases

Drug delivery via nanocarriers offers numerous advantages compared to traditional delivery methods. Nanoparticles (NPs) are composed of polymers, lipids, or metals and are less than 1 μm in size [1‒4]. The first generation of NPs-based drug delivery was developed primarily to enable higher therapeutic doses of drugs poorly soluble in water, in order to reduce toxic effects [5]. Since then, nanotechnology for drug delivery system has made considerable progress particularly in cancer therapeutics, diagnosis, and theragnosis. The strength of this nanotechnology is the possibility of modifying the physio-chemical parameters in the structure of the NPs to overcome the classic problems faced by most drugs. Specifically, NPs may protect the drugs from degradation and increase the bioavailability with consequent reduction in dosage. Surface modification on NPs may allow active targeting, reducing undesired off-target effects and therefore toxicity of drugs. Indeed, through modulations of main parameters of NPs including size, shape, solubility, morphology, surface charge, surface area, surface energy, as well as surface coating, NPs-based drug delivery has provided and still provides promising results for the site-specific delivery of drugs. In addition, targeted deliveries of anticancer drugs via nanocarriers may benefit from the enhanced permeability and retention effect, a pathophysiological phenomenon typical in solid tumors. This effect exploits the high blood supply of the tumor (permeability) due to the process of tumor angiogenesis and the lower ability of a substance present in the tumor mass to be drained by the lymphatic vessels (retention). Through enhanced permeability and retention effect, NP-based anticancer drugs could achieve higher accumulation in solid tumors relative to free drugs [6, 7].

Not surprisingly, the main government medicine regulatory agencies such as Food and Drug Administration (FDA) and European Medicines Agency (EMA) every year approve new therapeutic and diagnostic approaches based on NPs [8‒11]. Among these, an example is Onpattro, approved by the FDA in 2018 for the treatment of polyneuropathy caused by transthyretin-mediated hereditary amyloidosis or, even more recently, the approval of mRNA-based NPs for COVID-19 vaccination [10, 12, 13].

While most of significant progresses in nanomedicine have been mainly made in the field of cancer research so far, nanocarrier-based drug delivery approach has potential to apply for other major health problems including kidney diseases. Kidney is a fundamental organ for the maintenance of fluid, pH, and electrolytes homeostasis. Through the filtration of blood, kidney removes excessive fluid and waste products such as xenobiotics, endogenous metabolites, and drugs from our body by producing urine. Unrecognized kidney dysfunction due to gradual disease progression may often lead to imbalances of fluid and electrolytes, ultimately resulting in severe kidney damages. Common kidney diseases include glomerulonephritis, IgA nephropathy, nephronophthisis, polycystic kidney disease (PKD), acute kidney injury (AKI), and chronic kidney disease (CKD). In particular, more than 10% of world population have been affected by CKD, which is also commonly comorbid with other disorders including diabetes, hypertension, and cardiovascular diseases [14, 15]. While the impact of kidney diseases is increasingly recognized as a serious global health issue, the therapies for kidney diseases are still limited, partially due to the undesired side effects of currently used drugs.

In this review, we discuss the drug delivery systems via nanocarriers specifically targeting various kidney diseases. We first summarize the structural characteristics of the kidney, followed by the discussion on how the parameters such as size, charge, shape, material, and surface modifications of NPs affect the renal NPs’ biodistribution properties. Lastly, we wrap up by highlighting current developments in NPs therapy for kidney diseases.

A functional unit of the kidney, nephron, is composed of renal corpuscle and extending tubules [16]. In the renal corpuscle, the glomerulus, a bundle of capillaries, is contained within the Bowman’s capsule lined with outer layer of parietal epithelial cells. The glomerular filtrate is captured in the Bowman’s space and passed to the renal tubules for the reabsorption and secretion. The capillary wall forms the glomerular filtration barrier (GFB), a highly specialized structure for selective blood filtration. GFB is composed of three different types of cells: the luminal side of the capillary vessel is covered by fenestrated glomerular endothelial cells (GECs), which is attached to the second layer called glomerular basement membrane (GBM). Finally, the proximal component of GFB is visceral epithelial cells (podocytes) [17]. Since podocytes serve as the outermost aspect of the GBM, podocytes injury is often associated with proteinuria [18]. In addition, mesangium composed of mesangial cells and mesangial matrix is connected to GEC and provides a structural support and produces growth factors for the glomerular capillary network [19] (Fig. 1).

The characteristics of each component in this specialized filtering system are crucial for developing NP-based drug delivery approach targeting kidney. GEC lining the glomerular capillary lumen has direct contact with the blood and has fenestration ranging between 70 and 100 nm in diameter [20]. GBM is mainly composed of laminin, type IV collagen, nidogen, and heparan sulfate proteoglycan as observed in other basement membranes in the body. However, it is characterized with its exceptional thickness (300–350 nm) and particular components such as laminin-521, collagen α3α4α5(IV), and agrin. Their pore size is between 4 and 10 nm [21‒23]. Regularly interdigitated foot processes of podocyte form the filtration slit diaphragms with the size of 35–45 nm [24]. Taken together, the molecules smaller than 10 nm are generally reported to pass through the GFB [25]. In addition to the size selectivity, surface charge of GFB affects the filtration and reabsorption of small molecules by the kidney. The surfaces of GEC and podocytes are coated with gel-like layer called glycocalyx, which is rich in proteoglycans and glycosaminoglycans including heparan sulfate and hyaluronic acid [26]. Negatively charged heparan sulfate abundantly expressed in all three layers is the major contributor of the strong negativity of GFB to serve as the charge-selective barrier. Indeed, positively charged particles tend to cross the GFB more rapidly compared with anionic molecules due to electrostatic repulsion. It should be noted that both size and charge selectivity of GFB may be altered under the diseased conditions; thereby, larger molecules may abnormally cross the GFB [27].

The filtrates passing through the GFB are further proceeded in tubules for reabsorption into circulation via the peritubular capillaries, as well as for secretion to the urine to maintain the balance of fluids and electrolytes. Renal tubules are mainly composed of four segments, including proximal tubules, loop of Henle, distal convoluted tubules, and collecting ducts. Each segment consists of different types of cells specifically expressing specialized ion transporters or ion channels, thus possessing unique roles for the homeostasis. For example, proximal tubules play a fundamental role on the reabsorption of water, NaCl, NaHCO₃, glucose, amino acid, phosphate, as well as organic compounds [28]. This site is also critically involved in hormone production and metabolic functions. The major function of loop of Henle includes the reabsorption of water and NaCl [29]. The distal convoluted tubules located immediately after the macula densa participate in the transport of filtered NaCl, potassium, calcium, and magnesium [30]. Finally, the most distal part of the nephron, collecting duct, is the important site for the fine-tuning of water and salt reabsorption regulated by several factors including hormones, autocrine and paracrine factors, and osmotic conditions [31]. The consideration of these characteristics of nephron segments is the crucial aspect for the designing of NPs targeting kidney diseases. Below, we specifically discuss the influence of size, charge, shape, material, and surface modifications of NPs to target kidney diseases (Fig. 2).

The size of the NPs is one of the main parameters that influences their renal accumulation. NPs measuring <10 nm in size are generally associated with poor renal retention. Studies have demonstrated that they have a high clearance and after 24 h they are almost entirely removed in the urine [32, 33]. Interestingly, ultrasmall NPs close to the sub-nanometer range are retained in the kidneys for prolonged periods of time (>24 h), presenting a greater renal accumulation compared to those dimensionally close to 10 nm [34, 35]. A possible mechanism underlying this phenomenon might be that particles with a diameter ∼1 nm are retained inside the glycocalyx, which would therefore function as a molecular sieve [36]. Another intriguing feature concerns the NPs with sizes between 10 and 20 nm that pass through the GFB despite being slightly larger than the threshold size [37]. Among these, Wang et al. [35] demonstrated that kidney-targeted multimodal micelles with an average diameter of 15.0 ± 0.0 nm do not bind to any component of the GFB. In contrast, larger NPs (75 nm) have been reported to be retained by mesangial cells within the glomeruli and do not have access to tubular cells beyond the GFB [38]. Particles larger than 20 nm are too large to be filtered and present alternative strategies for reaching the various districts of the nephron. In particular, for those with an average diameter between 20 and 100 nm, a phenomenon of disassembly may occur [39]. For example, Zuckerman et al. [25] have described that while cationic cyclodextrin-containing polymer-based siRNA NPs (60–100 nm in diameter) do not disassemble in blood plasma, these siRNA NPs can accumulate and disassemble in GBM due to the abundant negative charge of proteoglycans present in GBM. NPs larger than 100 nm are generally unable to pass through the GFB but may reach the tubular components through distinct mechanisms similar to those for xenobiotics, endogenous metabolites, and drugs [40‒42]. Naumenko et al. [41] reported the renal accumulation and clearance of magnetic NPs (hydrodynamic diameter [HD] = 139 nm) through in vivo intravital imaging in mice. At an early stage, Cy5-labeled magnetic NPs were localized mainly in the basal compartment of the tubular epithelium, with subsequent increase in the lumen up to 40 min post-injection and gradual decrease due to excretion of magnetic NPs in urine (as demonstrated by urine iron concentration after cluster injection). These results suggested a mechanism for renal excretion of NPs larger than cutoff of GFB, involving the translocation of NPs from blood to urine via peritubular endothelial and renal epithelial cells. Other groups showed the similar mechanism with even larger NPs with diameter up to 400 nm. Deng et al. [40] demonstrated the kidney-targeted delivery of highly toxic and water-insoluble anti-inflammatory agent triptolide encapsulated in mesoscale NPs (∼400 nm). They suggested the delivery mechanism involving the endocytosis and exocytosis of endothelial cells of peritubular capillary, followed by the endocytosis of proximal tubule epithelial cells. A similar case reported by Williams et al. [42] demonstrated that mesoscale NPs conjugated to polyethylene glycol (PEG) with an average diameter of 400 nm were predominantly observed in the basolateral region of the proximal tubule epithelial cells. The amount of mesoscale NPs in the mesangial cells of the glomeruli was negligible, probably due to the pressure drop in the nephron and the large absorption pressure of the capillaries. Under normal physiological conditions, glomerular filtration cannot account for renal clearance of particles larger than 100 nm. Therefore, these findings are indicative of the presence of a tubular secretion mechanism. In summary, the size of the NPs is a variable that offers a lot of flexibility and versatility as by varying this parameter it is possible to target different renal cells.

Apart from particle size, another factor that influences renal reabsorption and filtration of NPs is their surface charge. As described above, the GFB is made up of three layers: two cellular layers (endothelial cells and podocyte) and a specialized extracellular matrix (GBM). The cellular layers are covered by a negatively charged gel-like coating, (glycocalyx) which, together with the anionic proteoglycans of the GBM, gives the GFB a strongly negative charge, behaving as a charge-selective barrier [43].

It was discovered that, depending on their surface charge, even NPs small enough to pass through the GFB interact with it differently and have distinct retention times. In particular, due to electrostatic repulsion the negatively charged NPs tend to flow through the GFB more slowly than positively charged ones, which have a faster ability to enter the tubular system [44]. In this regard, Liang et al. [45] evaluated the different distribution of oppositely charged quantum dots (QDs). After injection, the negatively charged QDs (HD = ∼3.7 nm, ζ = −52 mV) were initially distributed in the peritubular capillaries and were then gradually taken up by mesangial cells for up to 30 days, being detected only trace amounts in the urine. In contrast, cationic QDs of similar size but opposite charge (HD = ∼5.67 nm, ζ = +23.4 mV) were found to be readily excreted in the urine, with a renal retention rate 5 times greater than anionic QDs after 24 h (15%ID vs. 3%ID) [45].

Another mechanism in which surface charge influences the renal accumulation and clearance of NPs is their potential interaction with serum proteins, resulting in increased HD, thereby altering the rate of glomerular filtration [46]. Indeed, the mononuclear phagocytic system (MPS) represents one of the main obstacles that negatively influence the bioavailability of nanocarriers and therefore their tissue distribution. Made up of monocytes, dendritic cells, and macrophages, MPS sequesters NPs via opsonization and induces their degradation [47]. Although the PEGylation process is among the most used methods to obtain stealth nanocarriers, it has been demonstrated that the surface charge of the NPs also influences opsonization and therefore this parameter can be exploited to reduce their capture by the reticulum endothelial system, increasing the half-life of the particles [48]. Specifically, studies demonstrate that the use of NPs with neutral or zwitterionic surfaces may reduce opsonization with serum proteins including opsonins due to enhanced hydrophilic surface and anti-fouling properties and reduce the uptake through MPS in particular by liver [48]. Huang et al. [49] developed peptide amphiphile micelles functionalized with the zwitterionic peptide ligand (KKEEE)3K, which targets megalin receptor in proximal tubules [49]. The variation of the original peptide and the presence of additional zwitterionic peptide sequences led to the development of several NPs with different density and surface charge (zeta potential). Interestingly, even if all the micelles were localized in the kidneys, micelles consisting of 90% PEG₂₀₀₀-(KKEEE)3K (−41.4 ± 2.9 mV) had lower kidney accumulation than the positively charged (14.3 ± 1.6 mV) 90% PEG₂₀₀₀-(EEKKK)3E. Furthermore, micelles of negatively charged 90% PEG₂₀₀₀-(KKEEE)3K showed significantly enhanced liver uptake likely via MPS with respect to positively charged 90% PEG2000-(EEKKK)3E. Similarly, using mesoporous silica nanoparticles (MSNs) Droga et al. [50] analyzed the distribution of several functionalized MSNs. Positive charge with shielded surface amines (QA) correlates with greater urinary excretion compared to cationic MSNs with surface-exposed amines (PEI). This trend seems reasonable given that the glomerular capillary wall contains anionic charge, but it also emphasizes how crucial surface exposure of charged molecules is for influencing in vivo interactions. Therefore, these results show how the surface charge is a decisive parameter not only for determining renal clearance but also for biodistribution purposes and, in the same organ, for establishing tissue localization.

The shape of the particles is another parameter to take into consideration since it has a significant impact on the membrane adhesion process during the first phase of cellular internalization. While most of NPs have a spherical shape, recent improvements in nanofabrication techniques allowed the generation of NPs with different shapes possessing unique geometrical properties. These NPs may be classified according to their dimensionalities [51]. Spherical, dodecahedral, tetrahedral, octahedral, cubic NPs are among the 0-dimensional isotropic NPs. In contrast, nanotubes, nanoneedles, nanorods, or nanowires are classified as 1-dimensional, while plates or sheets-like NPs belong to the 2D shape class. Anisotropic 3D morphology of NPs are more complex, such as nanoflowers, nanostars, and polygonal nanoframes [51]. The mechanisms that lead to a variation in the half-life of NPs with different shapes are thought to be mainly their interaction with macrophages and the resulting membrane curvature [52]. Compared to high aspect ratio, NPs such as disk or rod-like NPs, spherical NPs have shorter circulation times. Indeed, the interaction of high aspect ratio NPs and macrophages with their one end may enhance the shear forces of blood flow on the free portions of NPs, thereby removing them from the cell surface prior to endocytosis and internalization. Furthermore, in contact with phagocytic cells, spherical NPs are susceptible to membrane curvature while this is unlikely to occur for high aspect ratio NPs. By circumventing these conditions, high aspect ratio NPs show longer half-lives and therefore a shape variation can be adopted to promote their renal accumulation [53]. Ruggiero et al. [54] demonstrated the renal clearance of single-walled carbon nanotubes with high aspect ratios and length (100–500 nm), although the molecular weight of these molecules is greater than the cutoff of glomerular filtration (30–50 kDa). It is likely that, the long axis of NPs can be increased by orders of magnitude without compromising the renal accumulation, as long as the width is within the cutoff diameter of GFB. In this way, high aspect ratio NPs may possess a great advantage as they could have an extended circulation time while retaining the kidney access. An example of the influence of shape on the renal clearance of NPs is provided by polyamidoamine dendrimers functionalized in one instance with PEG chains with a molecular weight of 2 kD, in the other with PEG with a higher molecular weight (20 kD) [55]. In the first condition, dendrimers (size of 18 nm) showed reduced renal excretion, accumulating predominantly in other organs. In contrast, in the latter case, the presence of PEG with a molecular weight of 20 kD gave the dendrimers a cylindrical structure (while maintaining the central core of 18 nm) and this led to an increase in their renal excretion. Furthermore, Rafidi et al. [56] reported the enhanced renal uptake and catabolism of nonbranched molecules with respect to branched molecules. These findings highlight the influence of molecule shape and hinge flexibility on the renal distribution and systemic exposure. Studies underlying the specific mechanisms that induce shape-dependent renal accumulation are currently limited. Given the initial results are encouraging, it is worth investigating the relationship between the geometry of the NPs and the site-specific delivery.

Nanocarriers for targeted drug delivery can be made from different materials including polymers, lipids, metals, carbon, silica, as well as proteins. The choice of material is critical since it determines the final properties of the nanocarrier and therefore its applications. Indeed, the material can influence factors such as pharmacokinetics and site specificity, but it can also aid in the encapsulation of the drugs, improving the efficiency in the formulation step. This is the case of liposomes that, with their lipid bilayer and an aqueous core, are suitable for the encapsulation of both hydrophobic and hydrophilic compounds. Polymers are among the most commonly used materials, how they allow for a controlled release of the drug. Proteins are also widely used, particularly because of their biocompatibility, while peptides are commonly employed to engineer nanocarriers and allow them to be directed site specifically [57].

In addition, nanomaterials have a different density and this is another parameter that can influence renal accumulation. For ultrasmall metal NPs (<8 nm), it has been shown that increasing the density of the material reduces renal clearance; therefore, these NPs will be eliminated by the kidneys more slowly and this may improve renal targeting [27]. According to Tang et al. [58], the density of the material affects the speed of the NPs to circulate in the bloodstream allowing NPs with lower density (<10 g/cm³) to circulate faster as they are located in the central region of the vessel where the speed is the highest. For instance, silica NPs (low density) [32] showed a 2-fold higher renal clearance compared to gold NPs (high density) [59], demonstrating that ultrasmall NPs with similar HD but different material density have different renal retention. More specifically, inorganic NPs exhibit lower renal clearance compared to organic ones, which are designed for greater retention at the renal cell level.

The surface functionalization process of NPs is carried out to improve their cellular uptake and biocompatibility through modifying physio-chemical and biodistribution properties. Actually, surface modification is also a tool to decrease the immune system response against NPs, reducing uptake by macrophages and, therefore, increasing their half-life [60, 61]. The addition of surface-exposed functional groups can be performed via two different approaches: covalent and noncovalent conjugation. Usually, covalent binding of ligands to the surface of NPs can be achieved using various linker molecules. Among these, the most common is PEG. Due to its polymeric nature, length and coverage density of PEG greatly influence the final characteristics of the nanocarrier. Indeed, Chithrani demonstrated that very short PEG molecules cannot prevent nonspecific protein adsorption, reducing the stealth effect of PEGylation compared to longer chains [62]. Furthermore, the presence of PEG prevents the aggregation of the NPs as it acts as a spacer, reducing the steric hindrance [63]. The non-covalent approach, mainly used for metallic and siliceous NPs, is based on a multitude of weak interactions such as electrostatic, van der Waals, hydrogen bonds and similar [64, 65]. An example of this type of system is the cucurbit[7]uril (CB[7]) functionalized gold nanorods (Au NRs) developed by Yue et al. [65]. Notably, the nanocarrier features modular non-covalent surface modifications with a molecule for targeted cancer therapy. Among weak interactions, sensitive bonding – such as heat or pH sensitivity – has also been extensively studied lately in order to design NPs for controlled drug release [66, 67]. In particular, the tumor microenvironment is very acidic compared to the normal microenvironment; pH-responsive nanoplatforms can be designed for controlled drug release triggered specifically by the acidity of the tumor environment [68, 69]. A model designed for this purpose, more distant from the traditional concept of NPs but equally effective, has recently been developed to precisely target the tumor by exploiting the pH variation. Yang et al. [70] designed a tumor-activated oncolytic peptide nanomachine that is automatically activated when entering the acidic tumor microenvironment by changing morphologically from NPs to nanofibrils. The restored α-helical structure of the nanofibrils binds to the membrane of tumor cells, inducing their lysis. This study was conducted in vitro and in vivo demonstrating tumor growth inhibition of 90% and 71%, respectively. Additionally, no in vivo off-target and hemolytic activities occurrences were discovered. Equally recent is the innovative delivery system made up of stellate MSNs loaded with lonidamine and polydopamine and coated with renal cell carcinoma (RCC) membranes (MLP@M) [71]. The external coating allowed specific targeting at the tumor level while irradiation with a near-infrared laser destroys the coating itself and the polydopamine leading to a dual effect: the rapid increase in the temperature of the tumor microenvironment above 45°C and the release of lonidamine (thermosensitive mitochondrial metabolism inhibitor) resulting in tumor cell apoptosis rate of 75%. In addition to the antiproliferative ability, this study also demonstrated a tumor targeting capacity of the nanocarrier associated with high biocompatibility.

Very often, despite the discovery of new drugs, their transition into clinical practice is complicated due to problems such as reduced bioavailability and/or the absence of specificity for the pathology of interest. For some of these drugs, such as chemotherapeutics, this specificity becomes fundamental to reduce damage to healthy cells, limiting toxic effects. In addition to the modulation of the physio-chemical parameters of the NPs for passive targeting, it is possible to functionalize the NPs to direct them toward a target site, carrying out what is called active targeting [72]. Although many molecules can be used as ligands, antibodies and peptides are among those mainly used for site-specific delivery to the kidney [35, 73]. Peptide ligands are widely used as they are naturally degradable, easily synthesized, customizable, and compatible with a variety of linkers. Furthermore, their small size (less than 50 amino acids in length) minimizes structural changes of the nanocarrier [74]. As previously described, Huang et al. [49] functionalized micelles with peptide 3K (KKEEE) targeting proximal convoluted tubule cells via the interaction between the peptide and the megalin receptor in the treatment of PKD. Numerous other peptides have been found to be effective in specifically targeting different regions of the nephron. Among these, the CYFQNCPRG peptide [75] and the CLPVASC peptide sequence are identified by Pasqualini [76] for targeted administration to the glomerulus, or the ELRGDMAAL peptide for drugs intended for the cortical collecting duct, for example, in the treatment of final stages of PKD [77]. Conjugation with antibodies is another strategy for precisely targeting NPs. Unlike peptides, antibodies are much larger molecules, with an average size of about 150 kDa. Therefore, while offering the advantage of site-specific delivery, they are bound to NPs that target tubular cells, so as to be secreted by the peritubular capillaries. Tietjen et al. [78] developed PLA-PEG NPs conjugated with anti-CD31 antibodies to target endothelial cells of the renal cortical vasculature. Using ex vivo normothermic machine perfusion, they found that the NPs vectored with anti-CD31 antibodies showed an accumulation in the renal vascular endothelial cells up to 10 times greater than the control NPs. Similarly, some researchers have studied the role of macrophages in rhabdomyolysis, a frequent complication of AKI [79]. To do so, they developed gold-coated iron oxide NPs carried by an anti-CD163 antibody that targets the CD136 membrane receptor expressed primarily by anti-inflammatory macrophages. Analysis of rhabdomyolysis-induced AKI on murine biopsies showed the presence of NP-CD163 within interstitial macrophages in the kidneys but not of NP-IgG (used as control).

In summary, the use of nanocarriers proves to be an extremely versatile and innovative tool, capable of protecting the drug from rapid degradation while also directing it. This leads to the development of increasingly precise and efficient NPs, created ad hoc for each specific purpose, thanks to the range of adjustments that may be implemented at the formulation level.

There are numerous diseases that affect the kidneys and, in most cases, those left untreated can lead to more serious conditions such as CKD or kidney cancer [80, 81]. To date, however, for many of the diseases affecting the kidneys, there is no definitive cure and this entails the need to search for new therapeutic strategies to improve the overall survival and quality of life of affected patients. Another problem is represented by drugs that cause nonspecific absorption and adverse side effects, compromising their use in clinical practice. In this context, a targeted drug delivery platform proves to be an effective tool to overcome the limitations of conventional delivery, providing a way to carry out specific targeting of drugs, reducing their systemic side effects but without lowering their therapeutic efficacy. The use of NPs in renal applications represents an expanding branch of medicine. Therefore, an increase in this trend can be expected in the coming years, also thanks to the numerous preclinical research showing promising results. In this regard, the most recent delivery systems applied to renal pathologies are described below and summarized in Table 1.

Glomerulonephritis is a rare kidney disease characterized by an inflammation of the glomeruli [93]. Glomerulonephritis is a complex pathology with many factors implicated in its onset and, to date, the pathogenesis is still unclear. Nonetheless, it appears that mesangial cells play a fundamental role in numerous glomerular diseases, including glomerulonephritis [94]. Not surprisingly, numerous research focuses on the delivery of molecules aimed at affecting mesangial cells. Between these, Liu et al. [82] developed cationic liposomes complexed with transforming growth factor-beta 1 (TGF-β1)-siRNA (lipoplex) coated with non-inhibitory plasminogen activator inhibitor 1R (PAI-1R). In vitro and in vivo results demonstrated the internalization of PAI-1R-Lip-siRNA into glomerular cells, with accumulation up to 6 h post-injection. The same result was not obtained in the case of liposomes without the target (Lip-siRNA), confirming the effectiveness of the PAI-1R peptide in cell-specific targeting. Furthermore, the release of TGFβ1-siRNA induced a significant reduction in gene expression, improving glomerulonephritis in the nephritic rat model. Zhang et al. [83] developed micelles for specific targeting of mesangial cells and macrophages, two major inflammatory cells associated with a vicious circle mechanism for glomerulonephritis progression. Dual targeting was mediated through the development of nanomicelles containing hyaluronic acid (HA) for phenotypic macrophage remodeling and dexamethasone (DXM) to inhibit abnormal proliferation of mesangial cells. The micelles were coated with endogenous macrophage membrane (MM), obtaining pH-sensitive biomimetic nanomicelles (MM/HA-DXM) capable of escaping phagocytosis and more easily reaching the glomerulus where the acidic microenvironment of glomerulonephritis induces the release of HA and DXM. In rat models of glomerulonephritis, it was demonstrated that the presence of the coating (MM) positively affects renal targeting compared to HA/DXM micelles, also confirming the reduced inflammation following the treatment and reduced levels of proteinuria (2.33 times) compared to control group. In another study, a co-delivery system for the therapy of immunoglobulin A nephropathy, the most frequent type of primary glomerulonephritis, was developed [84]. Specifically, they optimized nanoliposomes (110 nm) loaded with both p38α MAPK and p65 siRNA, two key inflammation-related proteins. Liposomes were then coated with the octa-arginine (R8) peptide, whose positive charge favors the interaction with the negative glomerular membrane, thereby increasing the retention and selective accumulation of NPs in the glomerulus, as demonstrated by the results obtained in vivo. Furthermore, in murine immunoglobulin A nephropathy models it has been demonstrated that the release of siRNA induces the silencing of the p38α MAPK and p65 genes, with consequent reduction of inflammation, proteinuria, and massive deposition of extracellular matrix.

There are many efforts aimed at achieving an effective therapy also for renal tumors. Among these, RCC is the most common malignancy with 14,000 deaths and 79,000 new cases each year in the USA [95]. Surgical excision is the gold-standard treatment for RCC but, where possible, is not always curative [96]. Furthermore, RCC is not very sensitive to radiotherapy and some pharmacological treatments can induce long-term resistance, leading to a reduction in their effectiveness [97, 98]. On the other hand, several studies have shown that some chemotherapeutics are able to sensitize cells to radiotherapy, leading to an ever-increasing interest in combined therapies [99]. Among these, Morais et al. [85] developed silver NPs (AgNPs) to enhance the effect of everolimus in clear-cell renal cell carcinoma and sensitize cells to radiotherapy. Specifically, compared to treatment with everolimus, the co-treatment with AgNPs + everolimus further reduced cell viability by approximately 20%. Furthermore, preliminary in vitro results showed that AgNPs would be capable of sensitizing cells making them more responsive to radiotherapy, most likely because both treatments act on the same target (mTOR), inducing apoptosis. Similarly, Rachamala et al. [86] reported the efficacy of liposomes loaded with everolimus and YM155, in order to sensitize RCC to radiotherapy via inhibition of mTOR and survivin, respectively. These liposomes showed significant inhibition of tumor growth both in vitro and in vivo, proving their greater efficacy compared to single treatments. The specificity of this delivery system is due to the conjugation of liposomes with a novel tumor-targeted peptide, as they previously demonstrated [100].

AKI is another frequent kidney disease, which leads to a rapid decline in kidney function, causing an increased risk of CKD and end-stage kidney disease [101‒104]. One of the main pathological mechanisms underlying AKI is the excess of reactive oxygen species, which induces a condition of oxidative stress [105]. To solve this problem, ultrasmall chitosan RuO₂ NPs (RuO₂NPs) have been developed, with the goal of removing excess reactive oxygen species while protecting tissues from oxidative stress and, therefore, having a significant therapeutic effect on AKI [87]. Experiments conducted in vivo on an AKI mouse model demonstrate that ultrasmall NPs have a notable cytoprotective effect on oxidative stress-induced nephrotoxicity. The catalytic properties of NPs mimic the activity of catalase, peroxidase, superoxide dismutase, and glutathione peroxidase, thus showing excellent antioxidant activity. In addition to their excellent therapeutic properties, NPs have a small size (≈2 nm) and this allows their rapid elimination, as confirmed by the low biological toxicity. In an even more recent study, cobaltosic oxide-polyethylene glycol-triphenylphosphine NPs were synthesized to exploit the properties of cobaltosic oxide in mitochondrial dysfunction, recently included among the underlying causes of the pathogenesis of AKI [88]. In particular, PEG and triphenylphosphine were included to improve biodistribution, increasing the half-life of the nanocarrier, and direct it to the mitochondria, as demonstrated by the accumulation of cobaltosic oxide-polyethylene glycol-triphenylphosphines in proximal tubule cells. On the other hand, cobaltosic oxide acts on mitochondria by inducing the expression of BNIP3 (responsible for mitophagy), alleviating AKI and inhibiting the progression of the disease toward CKD. Similarly, Peres et al. [89] developed gold NPs (AuNPs) for the treatment of subclinical AKI (subAKI), the early stages of kidney disease. The AuNPs switch the pro-inflammatory profile (response mediated by IFN-γ and IL-17) to the anti-inflammatory one (IL-4), demonstrating that their administration in subAKI mouse models prevents the tubule-interstitial injury characteristic of the disease, without nephrotoxicity in healthy mice.

Another interesting use of NPs is in peritoneal dialysis. Dialysis is one of the main treatment methods for patients with kidney failure [106]. Despite progress in treatment, the complications that can arise post-treatment (such as cardiovascular events and infections) are very serious; therefore, the mortality rate of patients undergoing dialysis remains very high [107‒109]. Furthermore, prolonged treatment periods can induce fibrosis of the peritoneal membrane, rendering peritoneal dialysis ineffective [110]. To reduce the side effects of peritoneal dialysis while maintaining its benefits, Silva et al. [90] have developed lipid core NPs carrying paclitaxel (LDE-PTX) as adjuvant therapy to peritoneal dialysis. Indeed, they demonstrated that treatment with LDE-PTX prevented the degeneration of the membrane function and its morphological changes, decreasing the expression of protein involved in fibrosis processes (α-SMA), the gene expression of pro-fibrotic markers (fibronectin and FSP-1), and the pro-angiogenesis factor VEGF. Finally, the encapsulation of PTX in LDE dramatically reduces drug-associated toxicity, but it may rather enhance its specificity to the cells with increased mitosis rates, which overexpress low-density lipoprotein (LDL) receptors. The advantage in the use of nanocarriers for the site-specific delivery of molecules in support of peritoneal dialysis is also demonstrated by other studies. For example, in previous in vitro studies Lee et al. [111] had demonstrated that their nanoliposome loaded with vitamin D3 had the same therapeutic effect as free vitamin D3. Therefore, in a subsequent study they tested vitamin D-loaded magnetic NPs in a mouse model of peritoneal fibrosis [91]. The nanocarriers were functionalized with an antibody (Ab-Vit. D-MNPs) directed against GPM6A, a peritoneal marker, to allow specific targeting. This study showed that the selective accumulation of NPs in the peritoneum has the same therapeutic effect as free vitamin D3 in improving peritoneal fibrosis. Importantly, Ab-Vit. D-MNPs ameliorate the side effects of overdose (hypercalcemia and body weight loss) in PD mice. Furthermore, in the field of peritoneal dialysis it has been demonstrated that nanotechnologies can also be used as a diagnostic method. Indeed, a study demonstrated how ferumoxytol, an iron oxide NP, can replace contrast agents in magnetic resonance imaging, paving the way for a new method for the treatment of arteriovenous fistulas [92]. The advantage of this nanosystem is the possibility of performing vascular mapping before creating hemodialysis access, minimizing unnecessary surgical procedures. Specifically, the comparative study (ClinicalTrials.gov: NCT02997046) on 59 participants with CKD highlighted that MR angiography with ferumoxytol predicted sections unsuitable for the creation of arteriovenous fistulas with greater success (11%) compared to the conventional duplex US, also showing excellent inter- and intrareader repeatability.

In this review, we described the potential of nanocarrier-based drug delivery systems applied to various kidney diseases. The optimization of nanocarrier characteristics such as size, charge, shape, and material may improve the targeted delivery of drugs in different cellular compartments of the kidney compared to free drugs. Nanocarriers can also be engineered to increase the site specificity, enhancing the effectiveness of treatment. Additionally, they have the potential to be used for diagnostic purposes in detecting kidney diseases at an early stage. On the other hand, although a number of studies show promising data, most of the studies on kidney-targeted drug delivery systems have been at the preclinical stage so far, performed on animal or cellular models. Therefore, there still remains several challenges to be addressed in order to translate the preclinical studies into patients. For example, the potential toxicity of nanocarriers in the long term has to be carefully evaluated to overcome the safety issues for the clinical use of nanocarrier-based drugs as NPs may have undesired side effects on respiratory, nervous, endocrine, immune, and reproductive systems [112]. The establishment of manufacturing process for the stable supply and quality standardization of nanocarriers may be another challenge for the practical use of nanocarrier-based drugs in the clinical settings. Further studies are thus required to fully optimize the properties of nanocarriers while ensuring their biosafety.

The authors have no conflicts of interest to declare.

Biogem, Molecular Biology and Genetics Research Institute, provided materials necessary for this study.

Laura Zucaro, Consiglia Longobardi, Antonio Miele, and Antonio Villanova wrote and reviewed the manuscript. Yoko Suzumoto designed, supervised, and revised the manuscript. All the authors read and agreed with the final version of the manuscript.

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.