Nanomedicine approaches for sirolimus delivery: a review of pharmaceutical properties and preclinical studies

Abstract

Sirolimus (rapamycin) is a mammalian target of rapamycin (mTOR) inhibitor with immunosuppressive, antiproliferative, antiangiogenic, antifungal, anti-restenosis and anti-inflammatory properties. However, its clinical application is often hampered by poor aqueous solubility, first-pass metabolism, transport by p-glycoprotein efflux pump, limited oral bioavailability and nonspecific distribution in off-target sites. Recently, various formulation strategies have emerged to overcome these limitations. Among these, pharmaceutical nanotechnology with numerous advantages has great potential for sirolimus delivery. Up to now, the only nanoparticle based FDA approved formulation in the market is Rapamune^® tablet which is composed of drug nanocrystals. This review focuses on recent studies that have been investigated various nanostructured carriers such as liposomes, micelles, polymeric nanoparticles, nanocrystals, magnetic nanoparticles, albumin nanoparticles, solid dispersion nanoparticles and niosomes for sirolimus delivery (in organ transplantation, cancer, vascular restenosis, etc.).

Graphical Abstract

Keywords:

• Sirolimus (rapamycin)
• nanocarriers
• liposomes
• micelles
• polymeric nanoparticles
• nanocrystals

Introduction

Sirolimus, also known as rapamycin, is a clinically important polyketide macrolide compound (Figure 1) originally produced by fermentation from Streptomyces hygroscopicus collected from Easter Island (Rapa Nui) [1]. The macrolactone ring of sirolimus is synthesized by mixed polyketide synthase/non-ribosomal peptide synthetase systems using 4,5-dihydrocyclohex-1-ene-carboxylic acid as a starter unit. The linear polyketide chain condensation with pipecolate and further cyclization form the macrolide ring that then undergoes a series of post-polyketide synthase modifications [2].

Figure 1. Schematic illustration of the mammalian target of rapamycin (mTOR) pathway and sirolimus (SIR) mechanism. mTOR integrates in/out from numerous upstream cues including several growth factors and interleukin-2 (IL-2). After entry into the cell, sirolimus associates with FK506 binding protein 12 (FKBP-12). The resulting complex interacts with the FKBP12-rapamycin binding domain located in the carboxyl terminus of mTOR and inhibits the mTOR kinase activity.

After entry into the cell, sirolimus associates with the abundant immunophilin, FK506 binding protein 12 (FKBP-12) (Figure 1). The resulting complex interacts with the FKBP12-rapamycin binding domain, located in the carboxyl terminus of mammalian target of rapamycin (mTOR) and inhibits the mTOR kinase activity [3]. By binding to and inhibiting mTOR, sirolimus blocks activation of p70S6 kinase resulting in cell cycle arrest at the G1 to S phase. mTOR inhibition also blocks interleukin-2 (IL-2) receptor and CD28-dependent signalling pathways. mTOR is the central component of a complex intracellular signalling pathway involved in cellular processes such as cell growth and proliferation, immunity, angiogenesis, fibrogenesis and metabolism (Figure 1) [4,5].

After oral administration of both liquid and solid formulations, sirolimus is rapidly absorbed with a time to peak blood concentration of about 1–2 h. It has poor (F = 14%) and variable oral absorption. First pass elimination by the intestinal and hepatic cytochrome P450 and p-glycoprotein activity likely contribute to low and variable bioavailability. Absorption is variably affected by food, especially high-fat meals that decrease the oral bioavailability [6]. Following absorption, most drug molecules in human whole blood, partition into red blood cells and approximately 40% of the drug in plasma is bound to proteins, mainly to albumin. Because of its lipophilic nature, sirolimus is widely distributed, displaying a large apparent volume of distribution (5.6–16.7 L/kg). Target whole blood sirolimus concentration ranges from 5 to 15 ng/mL. It is extensively metabolized by the cytochrome P450 isoenzyme CYP3A4 and hence extensive drug–drug interactions may occur [6,7]. Sirolimus biotransformation occurs by hydroxylation or demethylation and several metabolites have been identified. The terminal half-life has been reported to be in the range of 57–72 h. The majority of parent drug and its metabolites are excreted via the faeces, with only about 2% undergoing renal excretion [6,8]. Sirolimus pharmacokinetics displays wide inter- and intra-patient variability. Therefore, for prevention of graft rejection and minimizing toxicity, careful blood level monitoring is emphasized [9].

Isolated from fungi in the soil of Easter Island with anticandida activity, sirolimus subsequently shows impressive immunosuppressive activity. Demonstration of remarkable immunosuppressive potency in preclinical studies and clinical trials led to sirolimus approval for prophylaxis of renal graft rejection. It has been also investigated for liver, heart, lung and pancreas transplantation. Sirolimus is administered with other immunosuppressants (cyclosporine, corticosteroids, mycophenolate mofetil and tacrolimus) as early as possible after transplantation [3,10]. Sirolimus-eluting stents have been shown to be very effective in reducing coronary restenosis, re-intervention rates and other adverse cardiac events in patients with coronary artery disease [11,12]. Antiproliferative properties of sirolimus make it beneficial for the treatment of tumours and some benign pathological states associated with abnormal proliferation (Figure 2). Sirolimus and its analogues are beneficial for treating various other diseases and conditions, including tuberous sclerosis, Kaposi’s sarcoma, diabetes, lymphangioleiomyomatosis, uveoretinitis, keloids, scars, psoriasis, Muir–Torre syndrome and aging [13–15]. Approved and investigational uses of sirolimus are summarized in Figure 2 [13–27]. There are reports on radiosensitizing ability of sirolimus in different cancers [28–30]. Sirolimus is currently available as oral solution and tablet in market.

Figure 2. Indications for which sirolimus has been investigated. A growing number of evidence has demonstrated that sirolimus is beneficial for treating various diseases [13–27]. GVHD: Graft-versus-host disease; HIV: Human immunodeficiency virus; LAM: Lymphangioleiomyomatosis.

In spite of various promising pharmacological activities of sirolimus, it faces a number of limitations that need to be addressed by novel drug delivery systems. Sirolimus has some dose-dependent adverse effects, including immunosuppression, inhibition of bone growth, increased cholesterol, triglycerides and creatinine serum levels and decreased glomerular filtration rate. Leukopenia, thrombocytopenia, anaemia, rash, stomatitis, arthralgia, diarrhoea, hypertension and hypokalaemia may also occur [31]. Moreover, the lipophilic nature of sirolimus allows its wide distribution in lipid membranes resulting in a large volume of distribution and relatively long half-life [6,7].

Another major challenge for in vivo application of sirolimus is its strong hydrophobic nature which leads to difficulties in preparing intravenous formulations and delivery to a target site in effective concentrations. Sirolimus has a molecular weight of 914 g/mol, partition coefficient (log P) of 5.8, no ionizable functional groups within the pH range of 1–10 and a very poor water solubility (2.6 µg/mL) [32,33]. Poor water soluble drugs are generally dissolved using large amounts of excipients. However, the need for frequent drug administration raises more concerns due to possible toxic effects of the excipients. Sirolimus shows low oral bioavailability due to sensitivity to gastric acid, limited solubility and permeability, first-pass metabolism and transport by p-glycoprotein efflux pump [6,7].

High sequestration of free sirolimus into the erythrocytes may also hinder accessibility into target sites (tumour tissues). It shows inter- and intra-patient variability in drug pharmacokinetics [6,7]. The above mentioned limitations stress the need to improve delivery formulation of sirolimus.

The emergence of pharmaceutical nanotechnology has great potential for sirolimus delivery due to small size, enhanced drug solubilization and dissolution rates, high efficiency in drug encapsulation and cellular uptake, controlled drug release, cargo protection from harsh environment, escape from reticuloendothelial system (RES), long circulation residence, targeted delivery to specific areas and modulation of drug pharmacokinetics and biodistribution [34–36]. This review focuses on recent studies that have investigated various nanostructured carriers such as liposomes, micelles, polymeric nanoparticles, nanocrystals, magnetic nanoparticles, albumin nanoparticles, solid dispersion nanoparticles and niosomes for sirolimus delivery (in organ transplantation, cancer, vascular restenosis, etc.) (Figure 3).

Figure 3. Schematic representation of various nanocarriers for sirolimus delivery which are currently under in vitro and preclinical evaluation.

Sirolimus delivery using liposomes

Liposomes are spherical vesicles that have an internal aqueous phase surrounded by one or more bilayer membranes mimicking human cell membranes. They are considered the oldest nanoformulations and several liposomes have been approved in the last 20 years. Liposomes are composed of cholesterol and amphiphilic phospholipids. They are classified into uni-, oligo- or multi-lamellar according to number of layers and size. Depending upon the preparation methods and composition, size of liposomes ranges from around 50 nm to several microns [37–39]. Liposomes have several desirable properties for delivery of various therapeutic agents, including biodegradability, superior biocompatibility, sustained-release properties, capability of hydrophobic and hydrophilic drugs entrapment, low clearance rates, surface modification, safety, cargo protection from external degradation and flexibility in adjusting physicochemical characteristics [38,40]. They also preferentially accumulate at pathologic sites of inflammation, infections and tumours [41]. To shield from the recognition by RES and increase circulation time of liposomes, polyethylene glycol (PEG) conjugated phospholipids are inserted into the membrane bilayer and sterically stabilized (stealth) liposomes have been developed [42]. Moreover, by this modification conjugating different targeting moieties to the liposomal surface can be facilitated [43]. Many researches have been focused on sirolimus liposomal formulations and some recent examples have been summarized in Table 1.

Table 1. Nanocarrier compositions and characteristics of sirolimus liposomal formulations.

Nanocarrier composition	Size (nm)	Zeta potential (mv)	Entrapment efficiency (%)	Observations	Investigation status	Ref
DPPC, cholesterol, +/− DOPE	165–178	−41 to −40	73–77	Fusogenic pH-sensitive liposomes showed higher antiproliferative effects compared to conventional liposomes.	In vitro	[48]
DPPC, cholesterol, +/− PEG-poly (monomethylitaconate)- 6-(cholesteryloxycarbonyloxy) hexanol, +/− oleic acid	214– 240	−13 to −5	77–81	pH-sensitive liposomes improved selectivity and antiproliferative effect against cancer cell line.	In vitro	[49]
DPPC, cholesterol, DSPE-PEG and folate as targeting ligand	154	−13	88	Targeted theranostic thermal sensitive liposomes were suitable for tumour diagnostics as well as treatment.	In vitro/in vivo	[47]
PC, cholesterol	–	–	–	Liposomes improved graft survival, fibre outgrowth and neuroprotective effects.	In vitro/in vivo	[50]
SPC, cholesterol, +/− DSPE-PEG	155–181	−15–1	74–93	Antiproliferative activity against MCF-7.	In vitro	[51]
SPC, cholesterol, DSPE-PEG	124–137	−1–0	60–73	Co-loaded paclitaxel/sirolimus liposomes better controlled cancer growth.	In vitro/in vivo	[52]
EPC, cholesterol, DSPG, +/− DSPC	94–126	−8 to −5	73–98	Efficient inhibition of restenosis.	In vitro/in vivo	[44]
EPC, cholesterol, DSPG, DSPE-PEG	85–98	−11 to −8	99	Efficient inhibition of restenosis.	In vitro/in vivo	[53]
Phospholipids, cholesterol	150–500	–	–	Sirolimus liposomes were successfully prepared by rapid expansion of supercritical solution technology.	In vitro	[54]
PC, cholesterol, stearylamine/dicetylphosphate	100–165	Both positive and negative vesicles	–	Liposomes were not efficient for sirolimus solubilization.	In vitro	[55]
Lecithin, cholesterol	140–211	–	93–98	Liposomes increased tear production and were efficient for treatment of dry eye disease.	In vitro/in vivo	[56]

DOPE: dioleoylphosphatidylethanolamine; DPPC: dipalmitoylphosphatidylcholine; DSPC: distearoylphosphatidylcholine; DSPE-PEG: pegylated distearoylphosphatidylethanolamine; DSPG: distearoyl-sn-glycerophosphoglycerol; EPC: egg phosphatidylcholine; PC: phosphatidylcholine; SPC: soybean phosphatidylcholine.

In our series of studies on formulation of an effective drug delivery system for restenosis treatment, first we focused on preparation and optimization of sirolimus nanoliposomes [44]. Sirolimus liposomes were prepared by the remote film loading method. This method involved the addition of empty liposomes to drug thin film and the subsequent sonication for a few minutes. The effects of key formulation variables (cholesterol proportion, lipid to drug molar ratio, lipid composition and sonication time) on drug encapsulation efficiency (EE) and release rate were investigated using a fractional factorial design. By this approach, almost complete drug entrapment was achieved. In vitro release studies revealed that nanoliposomes could control the drug release for 3 weeks [44].

To develop the optimized carrier for local delivery, the effects of various physicochemical parameters of liposomes (surface charge, particle size and incorporation of PEG) on the anti-restenosis efficacy were evaluated using morphometric, immunohistochemical and in vivo computed tomography imaging analyses. The restenosis inhibitory efficacy decreased in the following order: cationic ∼100 nm vesicles ≥ cationic ∼60 nm vesicles > neutral ∼100 nm vesicles ≥ stealth ∼100 nm vesicles > anionic ∼100 nm vesicles [45]. Observing higher efficacy by 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) enriched cationic vesicles compared to other formulations, we proposed that more biocompatible chitosan coating could improve local retention and efficacy [46]. Sirolimus loaded chitosan decorated liposomes were prepared and characterized. In vivo evaluation by pathology, immunohistochemical and in vivo CT angiography imaging confirmed the greater advantages of chitosan dressed vesicles as nanocarriers for sirolimus intramural delivery over the conventional liposomes (Figure 4) [46].

Figure 4. Chitosan decorated sirolimus liposomes attenuated vascular restenosis following local delivery as confirmed by pathology, immunohistochemical and in vivo CT angiography. Reprinted from ref [46]. Copyright (2017), with permission from Elsevier.

In another study, sirolimus and indocyanine green (ICG) were co-encapsulated into folate targeted thermosensitive liposomes to enhance tumour therapeutic and diagnostic functions (Figure 5) [47]. ICG is a near infrared (NIR) fluorescent dye that generates high fluorescence and photoacoustic signals. The theranostic targeted thermosensitive liposome was ∼150 nm, spherical shape and stable for at least 4 weeks. Following irradiation, percentage of sirolimus released nearly doubled compared to without irradiation indicating that NIR-induced hyperthermia could accelerate drug release. Both NIR-induced hyperthermia and folate anchoring dramatically enhanced the cellular uptake of the two agents and cytotoxicity in HUVEC and HeLa cells. In vivo studies showed a much higher antitumour activity without causing systemic toxicity. Moreover, photothermal therapy and sirolimus showed synergistic effect on the ablation of tumour vessels (Figure 5) [47].

Figure 5. Schematic structure of the theranostic targeted thermosensitive liposome (A) and its antitumour mechanism (B). Redrawn with permission from Pang et al. [47]. Copyright (2016) American Chemical Society.

The proposed mechanisms for this efficient multifunctional carrier were as follow: (1) through enhanced permeability and retention (EPR) as well as folate mediated active targeting effects, the formulation was selectively accumulated at the tumour site and efficiently internalized into endothelial and tumour cells; (2) this theranostic probe precisely tracked the tumour tissue, focused the laser beam on the tumour region and converted NIR laser light energy to hyperthermia; (3) hyperthermia generated by activation of ICG under NIR irradiation mediated photothermal therapy and triggered sirolimus release from the thermosensitive liposomes; and (4) through blocking the regulation effect of mTOR by the released drug, antiproliferative and antiangiogenic effects were observed [47].

Sirolimus delivery using polymeric nanoparticles

Polymeric nanoparticles (NPs), invented in the 1970s, are solid colloidal systems composed of natural, synthetic or semi-synthetic polymers with size ranging from 10 to 1000 nm [57]. Chitosan, alginate and gelatine are a few examples of widely investigated natural polymers. Synthetic polymers extensively studied for various therapeutic delivery are poly (DL-lactide-co-glycolide) (PLGA), polycaprolactone (PCL), poly(lactic acid) (PLA), polyacrylic acid (PAA), poly(methyl methacrylates) and polyalkylcyanoacrylate [58,59]. Physicochemical properties of the employed polymer determine the fabrication process requirements and conditions to form NPs. Polymeric NPs have been extensively investigated as pharmaceutical delivery systems owing to their small size, controlled release properties, reduced toxicity, prolonged circulation time, cargo protection from harsh environments, enhanced therapeutics bioavailability, surface modification and functionalization and targeted capabilities [60]. Nanocapsules are reservoir-type NPs, in which the cargo is present in the core surrounded by a polymer membrane, while nanospheres are matrix based NPs in which the cargo is uniformly dispersed throughout the matrix [61]. Sterically stabilized (stealth) NPs, carrying PEG coatings, maintain an increased circulation time and predominantly increase the feasibility to accumulate in pathological sites due to EPR effect [42]. Table 2 summarizes the recent researches on sirolimus polymeric NPs.

Table 2. Nanocarrier compositions and characteristics of sirolimus polymeric nanoparticles.

Nanocarrier composition	Size (nm)	Zeta potential (mv)	Entrapmentefficiency (%)	Observations	Investigation status	Ref
PLGA	250	–	69	Periadventitial delivery of nanoparticles effectively reduced intimal hyperplasia.	In vitro/in vivo	[62]
PLGA	134–152	–	70<	Coencapsulation of sirolimus and a chemosensitizer (piperine) improved oral bioavailability of 4.8-folds.	In vitro/in vivo	[63]
PLGA, EGFR antibody as targeting ligand	274–287	−14 to −12	80 ± 4	Targeted nanoparticles showed superior antiproliferative activity over non-targeted nanoparticles and free drug.	In vitro	[64]
PLGA	180	–	88	Nanoparticles inhibited neointimal hyperplasia in vein graft without affecting the reendothelialization.	In vitro/in vivo	[65]
PLGA	307	–	80 ± 8	Nanoparticles enhanced sirolimus suppressive activity on dendritic cells and maintained an immunosuppressive profile in dendritic cells.	In vitro	[66,67]
PLA	233 ± 35	−30 to −20	–	Phagocytic activity of human polymorphonuclear neutrophils was promptly reduced but recovered. Therefore, they emerged as a robust cell species.	In vitro	[68]
Three nanoparticles: PLGA, PLA, Eudragit RS	180–280	–	–	Local delivery of sirolimus nanoparticles reduced neointimal growth. PLA formulation was the most efficient nanocarrier.	In vitro/in vivo	[69]
Chitosan/PLA	219–326	30	6–89	Sirolimus nanoparticles showed excellent immunosuppression in corneal transplantation.	In vitro/in vivo	[70]
Elastin-like polypeptides fused with FKBP12	4, 24	–	78, 99	Nanoparticles exerted potent therapeutic activity for Sjögren’s syndrome in a murine model.	In vitro/in vivo	[71]
Hyaluronic acid	10–11	−56 to −13	–	Hyaluronic acid nanoparticles improved pharmacokinetic profile of sirolimus and efficiently delivered drug to CD44-positive breast cancer cell.	In vitro/in vivo	[72]
N-isopropylacrylamide, methylmethacrylate, vinylpyrrolidone, acrylic acid	80 ± 34	−13 to −10	–	Oral delivery of nanoparticles robustly inhibited mammalian target of rapamycin pathway in a xenograft model of pancreatic cancer.	In vitro/in vivo	[73]
Poly(ε-caprolactone)-poly(ethylene glycol)-poly(ε-caprolactone)	170–180	∼0	84–100	High solubilization efficiency and sustained release profile were achieved by nanoparticles, proposed them as suitable carriers for immunosuppression in corneal transplantation.	In vitro	[74]
PEG200 end-capped poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)	207	–	92	Sustained delivery of sirolimus from nanocarriers enhanced cellular uptake and kinase inhibition efficiency.	In vitro	[75]
N-isopropylacrylamide, N-vinyl pyrrolidone, pegylated maleic polymer	54 ± 5	−8	84	Local delivery of nanoparticles to the injured artery inhibited intimal hyperplasia and promoted reendothelialization.	In vitro/in vivo	[76]

EGFR: epidermal growth factor receptor; FKBP12: FK506 binding protein; PEG: polyethylene glycol; PEG-PLA: polyethylene glycol-polylactic acid; PLA: polylactic acid; PLGA: poly lactic-co-glycolic acid.

Shi et al. [62] prepared and characterized sirolimus loaded PLGA NPs embedded in pluronic gel system for periadventitial delivery around the carotid artery immediately after open vascular reconstruction surgery. Sirolimus was encapsulated in NPs via a single emulsion method with respective EE and loading level of 69.1 and 11.6%. In vitro release studies revealed that drug loaded NPs dispersed in gel provided a more sustained release profile than free drug containing pluronic gel. Efficient uptake of NPs by vascular smooth muscle cells was observed in vitro, allowing for more sustained sirolimus delivery and durable inhibition of phosphorylation of S6 kinase. In vivo studies in a rat carotid injury model revealed that drug loaded NPs led to a more sustained inhibition of S6 kinase, vascular smooth muscle cells proliferation, intimal hyperplasia and restenosis compared to the free drug [62].

As we mentioned before, sirolimus low solubility and extensive efflux by p-glycoprotein limit its oral bioavailability. In a recent study, Katiyar et al. [63] reported PLGA NPs formulation of sirolimus, along with a chemosensitizer (piperine) for improved oral bioavailability and anti-cancer efficacy. In cytotoxicity studies on MDA-MB-231 cell line, half-maximal inhibitory concentration (IC50) of sirolimus solution and sirolimus NPs were 20.3 and 11.4 µM, respectively. By using everted gut sac technique, it was revealed that permeation of sirolimus was increased by NP formulation as well as by chemosensitizer treatment. After oral administration, 1.7-fold increase was observed in bioavailability of sirolimus suspension in the presence of the chemosensitizer suspension. Furthermore, there was a 3.5- fold increase in drug bioavailability of NPs as compared to drug suspension. The co-delivery of sirolimus and piperine by PLGA NPs resulted in ∼4.8-fold increase in bioavailability compared to drug suspension. This study has proven the potential of combination piperine/sirolimus NPs in the oral delivery of hydrophobic sirolimus [63].

Sirolimus delivery using micelles

Micelles are spherical or globular colloidal particles made of monolayers with diameters generally less than 100 nm. Traditional micelles, form from an average number (aggregation number) of surfactant monomers, generally have high critical micelle concentration (CMC) and low stability upon dilution. Recently, polymeric micelles, self-assembled NPs prepared from amphiphilic block copolymers in aqueous media, have attracted a great deal of attention due to lower CMC, better thermodynamic stability in physiological solutions, slower rate of dissociation allowing a longer drug retention time, narrow and small size distribution, higher solubilization capacity, controlled drug release and greater accumulation at the target sites mediated by escaping from RES uptake and EPR effect [77]. The block copolymer micelles can be subcategorized into at least two main types: 1) Amphiphilic block copolymer micelles and 2) polyion complex micelles or block ionomer complexes. A-B diblock copolymer, A-B-C triblock copolymer, grafted and hyperbranched polymers are different patterns of micelles formed by arranging these block copolymers [78,79]. Based on a series of studies, drug-loading capacity of micelles can be significantly improved by optimizing chemical structures of inner core for stronger carrier–cargo interaction [77]. Due to numerous advantages of micelles, many researches have been performed on micelles formulation and in vitro as well as in vivo characterization of sirolimus (Table 3).

Table 3. Nanocarrier compositions and characteristics of sirolimus micelles.

Nanocarrier composition	Size (nm)	Zeta potential (mv)	Entrapmentefficiency (%)	Observations	Investigation status	Ref
Methoxy poly(ethylene glycol)-poly(ε-caprolactone) block copolymer, +/− α-tocopherol	37, 101^a	–	90	Micelle formulations increased drug plasma concentration and decreased distribution into the brain.	In vitro/in vivo	[33]
Poly(ethylene glycol)-poly N-[N-(2-aminoethyl)-2-aminoethyl] aspartamide-b-poly(ε-caprolactone)	81–108	5–7	58–85	Sirolimus and siRNA micelles generated enhanced cytotoxicity and tumour regression in prostate cancer.	In vitro/in vivo	[83]
Poly(ethylene glycol)-block-poly(lactic acid)	37–44	–	–	Micelles containing paclitaxel, 17-AAG and sirolimus showed strong synergy in breast and lung cancer cells, potent synergistic anti-tumour activity in vivo and was well-tolerated after IV injection.	In vitro/in vivo	[80,81]
D-α-tocopheryl polyethylene glycol succinate	11	−4 ± 1	97 ± 1	Micelles significantly improved sirolimus oral absorption.	In vitro/in vivo	[84]
Methoxyl-poly(ethylene glycol)-succinic acid sirolimus conjugate	56, 94^b	−11	–	Combination of sirolimus and paclitaxel micelles improved potency against a multidrug resistant human breast cancer cell.	In vitro	[85]
Poly(ethylene glycol)-poly(propylene sulfide)	39 ± 1	–	41 ± 11	Sirolimus micelles prolonged skin allograft transplant survival.	In vitro/in vivo	[86]
Sulphated derivatives of Pluronic F-127 and poly(propylene sulfide)- poly(ethylene oxide) block copolymer	24	–	89 ± 10	Sulphate functionalized mixed micelles enhanced binding to collagen I coated surfaces, a potential for extracellular milieu binding.	In vitro	[87]
DSPE-PEG2000	14	−16 to −14	90–102	Phospholipid based micelles attenuated neointimal hyperplasia after balloon injury.	In vitro/in vivo	[53]
Amino-PEG–PE and N-palmitoyl homocysteine and cRGD as targeting ligand	10–15	–	–	Targeted micelles downregulated mouse cardiac endothelial cell as well as HUVEC production and inhibited release of pro-inflammatory cytokines.	In vitro	[88]
Cholesterol-polyethylene glycol	12–14	−1–0.4	77–82	Micelles showed a high scleral retention and permeation of sirolimus.	In vitro	[89]
O-octanoyl-chitosan-polyethylene glycol	41 ± 4	6.8 ± 0.3	86	Positively charged micelles showed a high scleral retention and permeation of sirolimus.	In vitro	[90]
Poly(ethylene glycol)-block-poly(2-methyl-2-benzoxycarbonyl-propylene carbonate)	66–76	–	15–88	Micelles significantly reduced sirolimus toxicity, but showed similar biodistribution profiles as drug nanosuspensions.	In vitro/in vivo	[91]
Pegylated octadecyl lithocholate and LTTHYKL peptide as targeting ligand	122–130	−1–0	88–91	Targeted micelle was introduced as a potential carrier for colorectal neoplasia with significantly less systemic toxicity and comparable therapeutic efficacy to free drug.	In vitro/in vivo	[92]

^a α-Tocopherol incorporation increased the micelle size to 101 nm.

^b Paclitaxel incorporation increased the micelle size to 94 nm.

17-AAG: 17-allylamino-17-demethoxygeldanamycin; Amino-PEG–PE: 1,2-diacyl-sn-glycero-3-phosphoethanol-amine-N-[amino-poly(ethylene glycol)]; DSPE-PEG: pegylated distearoylphosphatidylethanolamine; IV: intravenous.

Shin et al. [80] designed “Triolimus” a 3-in-1 poly(ethylene glycol)-block-poly(D,L-lactic acid) (PEG-b-PLA) based micellar nanocontainer for co-delivery of sirolimus, paclitaxel and 17-allylamino-17-demethoxygeldanamycin (17-AAG). The water solubility was increased by 80-, 10⁴- and 700- fold, respectively. All drugs were released simultaneously over 24 h period. 3-in-1 micelles had the highest cytotoxicity and combination index analysis revealed that 3-in-1 micelles exerted strong synergy in 4T1 and MCF-7 breast cancer cells. Acute toxicity of the micelle was surprisingly low at respective doses of 30, 60 and 60 mg/kg for sirolimus, paclitaxel and 17-AAG [80]. The investigators extended their initial observations and studied in vivo efficacy [81] as well as pharmacokinetics [82] of the nanosystem. Mechanistically, the drug combination inhibited both the PI3K/AKT/mTOR and Ras/Raf/MAPK pathways. In A549 and MDA-MB-231 tumour xenograft models, a three-infusion schedule of Triolimus displayed more potent and durable tumour control than paclitaxel-containing micelles [81]. After intravenous administration of Triolimus to mice at high doses of 30 mg/kg sirolimus, 60 mg/kg paclitaxel and 60 mg/kg 17-AAG, the area under the plasma concentration–time curves (AUC) of sirolimus and paclitaxel increased compared to single-drug‐loaded micelles. In contrast, following administration of the 3-in-1 micelles at modest dose (sirolimus, paclitaxel and 17-AAG at 5, 10 and 10 mg/kg, respectively), pharmacokinetic differences between 3-in-1 and single drug formulations were not observed [82]. All data suggested that the nano-package cocktail provided a novel and efficient approach for combination cancer therapy.

In another study, a trilayer polymeric micelle system composed of the triblock poly(ethylene glycol)-b-poly N-[N-(2-aminoethyl)-2-aminoethyl] aspartamide-b-poly(ε-caprolactone) (PEG-b-PAsp(DET)-b-PCL) polymer was synthesized and studied for co-delivery of siRNA targeting Y-box binding protein-1 and sirolimus [83]. The outer PEG layer was used to improve pharmacokinetics, the cationic pH responsive PAsp(DET) segment was designed to interact with siRNA, and the inner hydrophobic PCL compartment was used for efficient sirolimus entrapment. The co-loaded micelles were ∼108 nm and stable in phosphate buffered saline (PBS) for 7 d at room temperature. The micelles efficiently controlled sirolimus release rate. Combination siRNA/sirolimus micelles greatly enhanced in vitro cytotoxicity against PC3 prostate cancer cells and induced significant tumour regression and growth inhibition in a PC3 xenograft mouse model of human prostate cancer [83].

Sirolimus delivery using nanocrystals

One promising approach to overcome the solubility limitation of hydrophobic cargos is transforming the drug powder into nanocrystals or nanosuspensions. Nanocrystals are pure solid drug particles with a mean diameter below 1 µm and nanosuspensions are submicron colloidal aqueous dispersions of drug particles. Nanocrystals can be administered by various routes; however, product developments have mainly focused on the oral route of administration [93]. Particle size plays a key role in the in vivo performance of drug nanosuspensions. According to Noyes–Whitney equation (Equation (1)(1) $$\mathrm{dX}/\mathrm{dt}=\left(C\text{s}-C\text{t}\right)\times \mathrm{SD}/h$$(1) ), where dX/dt is the dissolution rate, S represents the surface area, D is the diffusion coefficient of the solute, h represents the thickness of the dissolution boundary layer, Cs is the saturation solubility and Ct is the bulk solution concentration, a decrease in particle size into the nanoscale causes an increase in total effective surface area, the dissolution rate and in vivo absorption [94,95]. (1) $$\mathrm{dX}/\mathrm{dt}=\left(C\text{s}-C\text{t}\right)\times \mathrm{SD}/h$$(1)

Bottom-up and top-down methods are the two primary techniques for drug nanocrystal production. The bottom-up processes involve the formation of NPs by controlled precipitation at nanometre scale, while the top-down processes associate with applying shear stress on large drug particles by milling or homogenization to reduce particle size [93,95,96].

Rapamune^® tablet was the first marketed nanocrystal product launched in 2002 by Wyeth Pharmaceuticals. Compared to drug solution, the tablet has the advantage of being easy to administer and 21% higher bioavailability. The oral single dose of Rapamune^® is 1 or 2 mg and the total tablet weight is ∼365 mg for 1 mg formulation and ∼370 mg for the 2 mg formulation. The inactive ingredients that can be found in the labelling information of the Rapamune^® are lactose, sucrose, PEG 8000, PEG 20,000, microcrystalline cellulose, calcium sulphate, pharmaceutical glaze, talc, magnesium stearate, titanium dioxide, poloxamer 188, povidone, glyceryl monooleate, dl-alpha tocopherol and carnauba wax. This product has been produced by the wet milling top-down method [96].

In this technique, drug particles are dispersed in a surfactant/stabilizer media and then particle size of the obtained suspension is reduced by milling energy. In NanoCrystal™ technology used in Rapamune^®, drug, dispersion medium, milling media and stabilizer are filled into the milling chamber. High shear force generated by the collision of the solid drug crystals with the milling media results in particle size reduction. The balls or pearls used as milling media consist of stainless steel, glass, ceramics or highly cross-linked polystyrene resin-coated beads [95,96].

Miscellaneous nanocarriers

Other nanoformulations have also been explored for sirolimus delivery such as magnetic NPs, albumin NPs, solid dispersion NPs, niosomes, nanoemulsions, β-cyclodextrin-based NPs and lipid-based NPs that are summarized in Table 4.

Table 4. Nanocarrier compositions and characteristics of sirolimus incorporated in miscellaneous nanoformulations.

Nanocarrier type	Nanocarrier composition	Size (nm)	Zeta potential(mv)	Entrapmentefficiency (%)	Observations	Investigation status	Ref
Magnetic nanoparticle	Glycerol monooleate coated Fe3O4, HER2 antibody as targeting ligand	130–275	∼30	∼95	Nanoparticles enhanced uptake and cytotoxicity in human breast carcinoma cell line.	In vitro	[99]
Magnetic nanoparticle	Fe3O4, mesoporous silica	∼80	−31	–	Dose-tunable co-delivery of hydrophobic and hydrophilic drugs (sirolimus–doxorubicin) improved tumour cell apoptosis and cytotoxicity.	In vitro	[100]
Perfuorocarbon nanoparticle emulsion	Perfuoro-15-crown-5 ether, phosphotidylcholine, phosphatidylethanolamine and glycerine	160–240	−15	–	Sirolimus nanoparticles improved skeletal muscle strength and cardiac contractile function efficiently targeting defective autophagy.	In vitro/in vivo	[101]
Solid self-microemulsifying drug delivery system	Capryol™ propylene glycol monocaprylate, glycofurol, TPGS, sucroester 15 and mannitol	108 ± 11	–	–	The formulation showed enhanced release rate, higher stability in acidic pH and improved oral bioavailability.	In vitro/in vivo	[102]
Nab-Rapamycin	Albumin protein	∼100	–	–	Adventitial injection of Nab-Rapamycin reduced restenosis in a porcine model by immunosuppressive and antiproliferative mechanism.	In vivo	[103]
Solid dispersion nanoparticle	PVP K30	∼250	–	–	Solid dispersion nanoparticles prepared by a supercritical antisolvent process enhanced sirolimus oral bioavailability.	In vitro/in vivo	[104]
Nanoparticle	PVP K90, SDS	∼30	–	–	The sirolimus nanoparticles prepared by a continuous flow method resulted in faster and improved oral absorption.	In vitro/in vivo	[105]
Niosome	Span 60, cholesterol, +/− PEG-poly (monomethylitaconate)- 6-(cholesteryloxycarbonyloxy) hexanol, +/− CHEMS	268–305	−16 to −11	63–71	pH-sensitive niosomes showed higher antiproliferative effects compared to conventional niosomes.	In vitro	[98]
Nanoemulsion	Traicetin, Tween 80, Tween 20, iso-propanol, PEG 400, propylene glycol, Labrasol, Cremophor RH 40	14–47	0–1	–	Oil-in-water Triacetin mediated nanoemulsions showed cytotoxicity against cancer cells and improved permeability across Caco2 cells.	In vitro	[106]
β-cyclodextrin based nanoparticle	Acetalated β-cyclodextrin	185–250	–	90<	Sustained release nanocarriers improved antiatherosclerotic activity of sirolimus.	In vitro/in vivo	[97]
Lipid-polyaniline nanoparticles	Emeraldine base polyaniline, DPPC, DSPE-PEG and folate as targeting ligand	141	−0.3	–	Imaging guided combinational photothermal and chemo cancer therapy.	In vitro/in vivo	[107]
SAINT-O-Somes	DSPE-PEG, SAINT-C-18, POPC, cholesterol and anti-human VCAM-1 as targeting ligand	128 ± 4	11 ± 1	73	Targeted SAINT-O-Somes efficiently inhibited AB8/13 cells migration and proposed as drug delivery vehicle to podocytes.	In vitro	[108]

CHEMS: cholesteryl hemisuccinate; DPPC: dipalmitoylphosphatidylcholine; DSPE-PEG: pegylated distearoylphosphatidylethanolamine; Nab: nanoparticle albumin-bound; PEG: polyethylene glycol; POPC: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; PVP: polyvinylpyrrolidone; SAINT-C-18: 1-methyl-4-(cis-9-dioleyl)methyl-pyridinium-chloride; SDS: sodium dodecyl sulphate; TPGS: D-α-tocopheryl polyethylene glycol 1000 succinate; VCAM-1: vascular cell adhesion molecule-1.

In a recent study, Dou et al. [97] developed an acetalated β-cyclodextrin (Ac-bCD) based NP for sustained sirolimus delivery for atherosclerosis (Figure 6). For chronic inflammatory diseases, biodegradable materials (such as polyanhydrides and polyesters) with acidic byproducts may lead to local inflammation. In this study, kinetically controlled acetylation was used to synthetize biocompatible and biodegradable materials of Ac-bCDs. Ac-bCDs degrade into a non-acidic and water-soluble parent compound under physiological conditions. High EE (>90%) and slowed release rate were achieved. Pharmacokinetic studies revealed a prolonged profile of drug concentration in the blood after subcutaneous administration of 3 mg/kg sirolimus NPs. Nearly constant drug levels in the blood and the aorta (around 50 ng/L and 1.2 ng/g, respectively) were maintained for 13 d (Figure 6(B)). Apolipoprotein E-deficient mice were treated by subcutaneous administration of drug NPs at the lower (1.0 mg/kg of sirolimus) and higher (3.0 mg/kg of sirolimus) dose every 15 d. The average lesion area was 4.7 and 3.9%, respectively (Figure 6(C)). Sirolimus treatment significantly diminished macrophages and matrix metallopeptidase 9 (MMP-9) in the plaques and serum inflammatory cytokines including tumour necrosis factor α (TNF-α), interferon-γ (INF-γ), IL-1α and IL-1β. Moreover, NPs treatment dramatically stabilized atherosclerotic plaques. These effects were more remarkable for NPs treatment groups as compared to the free drug group. These findings demonstrated that sustained sirolimus delivery may provide more significant antiatherosclerotic activity over traditional administration through selective inhibiting of mTORC1 signalling in apolipoprotein E-deficient mice [97].

Figure 6. Design of sustained sirolimus delivery nanomedicines based on acetalated β-CD (A), sirolimus concentrations in the blood and the aorta of C57BL/6 mice (n = 4, mean ± SEM), after subcutaneous administration of sirolimus nanoparticles at 3 mg/kg of drug (B). Therapy with sirolimus nanoparticle significantly reduced atherosclerosis in ApoE−/−mice (C). (C) Representative photographs of total aortas from each group and quantitative results subjected to various treatments. Reprinted with minor modification from Dou et al. [97]. Copyright (2016), with permission from Elsevier.

In another study, pH-sensitive niosomal formulation was prepared and characterized to improve sirolimus efficacy and selectivity [98]. Vesicles were composed of Span 60 and either cholesteryl hemisuccinate (CHEMS) or PEG-Poly (monomethyl itaconate)-CholC6 copolymers for pH sensitivity and prepared by a modified ethanol injection method. Vesicles’ physicochemical characteristics, pH-sensitivity, stability and in vitro antiproliferative activity were evaluated. Mean particle size of formulations was ∼285 nm and %EE of prepared niosomes was in the range of 68.2–75.3%. Drug release rate from two pH-sensitive formulations at pH 7.4 was much slower than release rate at pH 6.5. However, after 1 h incubation of CHEMS-based vesicles in plasma, pH-sensitivity property was lost and high release rate occurred at pH 7.4. Whereas, pH-sensitive PEG-Poly (monomethyl itaconate)-CholC6 bearing vesicles were stable in plasma and could release drug in the mild acidic environments, often present in endosomal compartment and tumour. Higher antiproliferative activity against erythromyeloblastoid leukaemia cell line was observed with both pH-sensitive niosomes compared with conventional niosomes. In contrast, conventional niosomes showed higher antiproliferative effect on HUVEC cells as a healthy cell line. The results revealed that this novel pH-sensitive niosome can improve sirolimus in vitro efficiency [98].

Conclusions

The preclinical and clinical studies performed over the last two decades have shown that sirolimus (rapamycin) shows efficacy in different diseases and conditions, including prophylaxis of graft rejection, restenosis, tumours, Kaposi’s sarcoma, diabetes, lymphangioleiomyomatosis, uveoretinitis, keloids, scars, psoriasis and aging. Although sirolimus has shown efficacy in various pathological conditions, its potency has substantially diminished by sirolimus dose-dependent adverse effects, wide distribution into lipid membranes, extensive apparent volume of distribution, strong hydrophobic nature, difficulties in preparing intravenous formulations, low oral bioavailability, high sequestration of free drug into the erythrocytes, low accessibility into target sites and inter- and intra-patient variability in drug pharmacokinetics.

To overcome these obstacles, the various nanocarriers, including liposomes, micelles, polymeric NPs, nanocrystals, magnetic NPs, albumin NPs, solid dispersion NPs, nanoemulsions, lipidic NPs and niosomes have been under development and evaluation. As summarized in this article, many of these nanoformulations have been found to be appropriate for the sirolimus incorporation as well as controlled and targeted sirolimus delivery to improve its effects in several distinct diseases and reduce its side effects. The characteristics of these nanosystems can be tuned according to the specific requirements of the target disease. Based on our understanding from the literature, the growing number of sirolimus researches in chemotherapy for cancer treatment can improve existing therapies by targeting tumours as well as by reducing the administered dose.

Impressive results of different sirolimus nanostructures and improved drug efficacy in the cell lines and animal models highlight their potential for evaluation in clinical trials to establish their effectiveness in clinics. In spite of numerous advantages of the studied nanocarriers, the main obstacles are lack of tissue and cell specificity and off-target drug distribution. A few studies were focused on combination of sirolimus with contrast-, imaging-, antibody- or ligand-mediated targeted delivery. Therefore, future studies are greatly needed to achieve better-targeted and advanced therapeutic modalities. Furthermore, more attention should be paid to the toxicity of sirolimus nanoformulations, particularly after repeated and high dose administration. So far, the emphasis of most sirolimus nanoformulations is particularly on treatment of cancer and vascular restenosis. Studies on potential of these nanocarriers for treatment of other conditions, including diabetes, Alzheimer, infections, keloids, scars, psoriasis and aging are in great demand.

Abbreviations
17-AAG	=	17-allylamino-17-demethoxygeldanamycin
Ac-bCD	=	acetalated β-cyclodextrin
AUC	=	area under the plasma concentration–time curves
CHEMS	=	cholesteryl hemisuccinate
CMC	=	critical micelle concentration
DOTAP	=	1,2-dioleoyl-3-trimethylammonium-propane
EE	=	encapsulation efficiency
EPR	=	enhanced permeability and retention
FKBP-12	=	FK506 binding protein 12
GVHD	=	graft-versus-host disease
HIV	=	human immunodeficiency virus
HUVEC	=	human umbilical vein endothelial cells
IC50	=	half-maximal inhibitory concentration
ICG	=	indocyanine green
INF-γ	=	interferon-γ
IL	=	interleukin
LAM	=	lymphangioleiomyomatosis
mTOR	=	mammalian target of rapamycin
MMP-9	=	matrix metallopeptidase 9
NIR	=	near infrared
NPs	=	nanoparticles
PAA	=	polyacrylic acid
PBS	=	phosphate buffered saline
PCL	=	polycaprolactone
PEG	=	polyethylene glycol
PEG-b-PAsp(DET)-b-PCL	=	poly(ethylene glycol)-b-poly N-[N-(2-aminoethyl)-2-aminoethyl] aspartamide-b-poly(ε-caprolactone)
PEG-b-PLA	=	poly(ethylene glycol)-block-poly(D,L-lactic acid)
PLA	=	poly(lactic acid)
PLGA	=	poly (DL-lactide-co-glycolide)
RES	=	reticuloendothelial system
SIR	=	sirolimus
TNF-α	=	tumour necrosis factor α

Disclosure statement

No potential conflict of interest was reported by the authors.