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Folate appended cyclodextrins for drug, DNA, and siRNA delivery
Accepted Manuscript Folate appended cyclodextrins for drug, DNA, and siRNA delivery Magdalena Ceborska PII: DOI: Reference:
S0939-6411(17)30490-3 http://dx.doi.org/10.1016/j.ejpb.2017.09.005 EJPB 12590
To appear in:
European Journal of Pharmaceutics and Biopharmaceutics
Received Date: Revised Date: Accepted Date:
18 April 2017 1 September 2017 8 September 2017
Please cite this article as: M. Ceborska, Folate appended cyclodextrins for drug, DNA, and siRNA delivery, European Journal of Pharmaceutics and Biopharmaceutics (2017), doi: http://dx.doi.org/10.1016/j.ejpb. 2017.09.005
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Folate appended cyclodextrins for drug, DNA, and siRNA delivery Magdalena Ceborska*[a] ORCID 0000-0001-5555-771X
Institute of Physical Chemistry, Polish Academy of Sciences Kasprzaka 44/52, 01-224, Warsaw, Poland E-mail: [email protected]
Abstract Drug, DNA, and siRNA delivery systems based on cyclodextrin (CD) core and connected with folate (FA) via various linkers are presented. They include simple monoderivatized cyclodextrins as well as cyclodextrins with higher degree of substitution, both in their primary and secondary sides. Examples of simple polymers and dendrimers are also discussed. Such carriers possess properties inherent to both of their components. Cyclodextrin provides the ability to encapsulate organic molecules in its inner cavity, thus improving their solubility in water, bioavailability, and stability, while FA assures targeting folate receptor overexpressing cancer cells. Drug delivery systems loaded with drugs such as e.g. methotrexate, doxorubicin, paclitaxel, vinblastine, and docetaxel were found to have superior properties as compared to the free drug. Dendritic folate appended cyclodextrins were also found to be good sustained release systems for DNA and siRNA. The recent progress in the synthesis and drug, DNA, and siRNA delivery application of folate appended cyclodextrins is presented.
Keywords: cyclodextrin • folic acid • bioconjugate • drug delivery
1
1. Introduction Living cells depend upon vitamins for their proper function while cancer cells need them supplied in larger quantities for rapid growth sustenance. As vitamin uptake receptors (VUR) are often overexpressed on the surface of cancer cells, folate receptor (FR) is broadly utilized for tumor cells detection as well as for cancer-targeted drug delivery. There are two main strategies designed for targeted drug delivery to FR overexpressing tumor cells – first is a coupling of the desired drug to the monoclonal antibody against the FR, second a conjugation with folic acid (FA, Figure 1a) which shows high affinity to FR and thus good targeting properties [1,2]. FA (vitamin B9) is a small molecule built from p-aminobenzoate linked to a pteridine ring, and glutamic acid [3]. It possesses in its structure functionalities that allow for a FA conjugation to various molecules in order to form specific prodrugs. Conjugation via g-carboxyl group is preferred as it has no impact on the FA-receptor binding properties [4]. There is a great number of folate-based carriers described in the literature, including micelles [5], nanoparticles [6], liposome-folate-encapsulated drugs [7], and cytotoxic drugs-folate conjugates [8-13]. To improve the water solubility of drugs, encapsulation of bio-relevant molecules in cyclodextrins may be applied. To achieve both: tumor targeting properties and ability to increase water solubility, targeted drug delivery systems built from cyclodextrin covalently bonded with folate, are used. Native cyclodextrins (a-CD, b-CD, and g-CD) are macrocyclic oligosaccharides with a hydrophobic cavity and a hydrophilic external surface, which makes them readily soluble in water [14]. Their ability to form inclusion complexes with various organic molecules in solution [15] and in the solid state [16] is utilized by cosmetic [17] and food [18] industries. Derivatization of CDs leading to amphiphilic molecules increases their interactions with biological membranes, thus making them suitable for biomedical applications.[19] Their good water solubility combined with low immunogenicity [20] enables their broad application in pharmaceutical industry [21,22]. This paper features the recent progress in the synthesis and drug delivery application of folate appended cyclodextrins. Comparison of the folate-appended cyclodextrins with other FA targeted drug-carriers including simple folate, liposome-folate conjugates, and folatedecorated nanoparticles is also presented.
2
Figure 1.a) Folic acid b) native (a-, b-, and g-) cyclodextrins.
2. Mechanism of the folate mediated delivery of drugs to FR-a positive cancer cells. Folate is delivered into cells via the reduced folate carrier [23], the proton coupled folate transporter [24] or the folate receptor, which exists in four isoforms (FR-a, FR-b, FR-g, and FR-d) [25,26]. FR-a and FR-b are membrane-associated proteins anchored to a glycosylphosphatidylinositol (GPI) membrane. FR-a exhibits higher than FR-b affinity towards 5-methyltetrafolate. It is mainly expressed on the apical surfaces of epithelial cells [27,28], while FR-β is expressed on macrophages [29] and monocytes [30]. FR-γ was identified as a secretory protein from hematopoietic tissues and is characterized by much lower affinity towards folate than folate receptors FR-a and FR-b. The FR-d, which is hard to detect in human tissues, was only found on regulatory T cells [31]. The mostly studied FR-a is expressed at very low levels in normal tissues, but is overexpressed in many types of cancer. It is due to high demand of folate by rapidly dividing cells. Therefore, FR-a is utilized both as tumor cell marker, as well as the target for delivery of imaging and therapeutic agents [32,33]. The possible mechanism of targeted drug delivery via FA-CD conjugate/drug supramolecular complex is analogous to that of FA-drug covalent conjugate [34]. In a FA-drug conjugate drug molecule is linked to FA by easy to cleave linker (e.g. with disulfide bonds, Figure 2a), while in FA-CD conjugate/drug supramolecular complex it is kept in CD cavity by
3
weak interactions and hydrophobic forces (Figure 2b). The mechanism of the drug delivery to the FR-a positive cancer cells depends on the receptor-based endocytosis (Figure 2). The FA part of the drug delivery system ensures targeting of a folate receptor. Subsequently the FA-drug covalent conjugate (or FA-CD conjugate/drug supramolecular complex) enters endosome, where the drug is cleaved, which is followed by the free drug leaving the endosome to act upon cancer cells.
Figure 2. Mechanism of a folate-mediated delivery of drugs to FR-a positive cancer cells via receptor-mediated endocytosis a) using FA-drug conjugate b) using FA-CD/drug host: guest complex.
3. Folate-appended cyclodextrins as drug, DNA and siRNA delivery systems. The drug delivery systems based on cyclodextrins rely mainly on the formation of supramolecular host : guest inclusion complexes, where the bio-relevant molecule is wholly or partially encapsulated inside the macrocyclic cavity. While designing new potential receptors with cyclodextrin core, derivatized with various substituents, one has to bear in mind that covalently linked substituents may also penetrate the CD’s cavity thus hindering the inclusion of the guest molecule [35]. It is known that native (a-, b-, and g-) CDs are able to form associates with FA [36-38]. a-CD forms with FA weakly bound exclusion compound where guest molecule interacts with the secondary hydroxyls located at the wider rim of a-CD. band g-CDs form with FA pseudorotaxane structures, where the host molecule is threaded over
4
the guest FA. Therefore, to incorporate folate-appended CDs into the specific drug delivery systems, the requirement of the formation of stronger CD/drug than CD/FA complex should be fulfilled. In the present paper the recent progress in the synthesis and drug delivery application of folate appended cyclodextrins is presented. It can be clearly seen that b-CD is by far the most utilized cyclodextrin, with only a few reported examples of FA derivatization of a- and g-CDs. In Figure 3, drug molecules complexed with CD-FA conjugates described in this review are presented. The main features of the complexes (method of preparation, stoichiometry, and values of association constants) are presented in Table 1 presented at the end of chapter 3 (only for the complexes where such data was reported). Whenever the mode of complexation was described it is also noted in the main text. Moreover, Table 2 presented at the end of chapter 4 gives a simplified information about the results of application of specific FAappended-CD/drug complex for specific tumors.
5
Figure 3. Targeted drugs: methotrexate (MTX, 1), scutellarin (SCTL, 2) b-estradiol (b-EST, 3) chlorambucil (CLBL, 4), 5-fluorouracil (5-FU, 5), doxorubicin (DOX, 6), paclitaxel (PCX, 7), carboplatin (CBP, 8). 3. 1. Simple substituted cyclodextrins and polymers. 3.1.1. Cyclodextrin - folic acid - amido conjugates. Clementi et al [39] first synthesized the b-CD-FA conjugate, where FA was linked to 6-[2-aminoethyl)amino]-6-deoxy-b-CD (EN-b-CD-FA) via FA carboxyl groups (a and g) to form amido derivatives (Figure 4, only g- isomer is shown). The synthetic route, enabling formation of only desirable g-isomer, was later improved by Tofzikovskaya et al [40]. The proposed receptor increases significantly the photostability of FA, thus enabling its broader and easier applications, e.g. in food industry. Later, the cytotoxity of EN-b-CD-FA was evaluated [41] using MTT assay, MCF-7 (breast), BEAS-23B (normal lung), A5649 (lung cancer), and HeLa(cervical) cell lines. EN-b-CD-FA showed no cytotoxicity towards the studied cell lines rendering it safe for medical use.
Figure 4. Synthesis of CD-FA g-amido-conjugate. The 1:1 host : guest inclusion complex with antifolate cytotoxic drug methotrexate (MTX) was synthesized. It exhibited lower (four times) cytotoxity towards normal cell lines and higher cytotoxity towards all of studied cancer lines (as compared to free MTX). Another drug subjected to FA-appended CD was scutellarin, flavone used in the inhibition of platelet aggregation and for the improvement of blood circulation [42]. To increase its low water solubility as well as bioavailability it was reacted with three polyamine b-CD-FA conjugates:
ethylenediamine-b-CD-FA(EN-b-CD-FA),
6
diethylamine-b-CD-FA
(DETA-b-CD-FA) and triethylamine-b-CD-FA (TETA-b-CD-FA) [43] giving 1:1 inclusion complexes. In all three complexes scuttelarin enters CDs with cavity through its wider rim. Upon formation of host : guest complex the solubility of scutellarin increased significantly (approximately 1.2*102, 2.3*102 and 3.1*102, respectively). Xu et al [44] used the CD-FA conjugate in which b-CD is linked by a g-carboxyl group of folic acid with the amino group of 6-monodeoxy-6-(2-aminoethyl)-β-CD for the delivery of docetaxel (DCX) Figure 7), a drug used in treatment of breast, lung, and prostate cancers. 1:1 Complex of DCX with CD-FA conjugate exhibited significant antitumor efficacy (leading to cell apoptosis of folate receptor cancer cells). 3.1.2. Cyclodextrin - folic acid conjugates linked by polythylenimine and polyethylene glycol spacers of various lengths. Tang et al [45] developed a folate-polythylenimine-cyclodextrin (CD-PEI-FA) polymer which both in vitro and in vivo proved to be efficient gene delivery system which may be used in cancer gene therapy (Figure 5). Polyethylenimines (PEI) of various lengths are characterized by good transfection efficiency in the wide range of cells and are known to form polyplexes with DNA. The obtained FA-PEI-CD polymer allowed for a valuable transfection of broad array of cancer cells with low cytotoxicity.
b-CD 1)CDI b-CD 2) PEI-600, Et3N H N
N H
N H m1
FA
m2
CDI, NEt3N
b-CD
N H
H N
N H m1
O
O
H N m2
O O
N
n
O N H
O N H
HO O
Figure 5. Synthesis of CD- PEI-FA.
7
H N
O N N
NH N
O
O
H N
NH2
N H
O O
n
Folate terminated polyrotaxanes (FPP) consisted of a-CD decorated with PEI600 threaded over PEG chained capped with FA molecules at both sides were prepared (Figure 6) [46]. FPP/pDNA polyplexes were obtained and were shown to possess low cytotoxicity and high efficiency of DNA delivery to target cells.
a-CD
O H2N
O
O
NH2
m
H2N
O m
NH2
X
FA-NHS O O N H2N
H N
N N
O
O
N H
N H
N
O O m
O
X
O
N H
O
O
N H
O
H N
N N
N N
O
H2 N
N
CDI
H N
NH2
N m1 H
m2
O N
NH2
O
H N
N
N H2N
N
N
H N
H N
N
N H
N
N
N
NH2
NH2 O
O O
NH2
n O
O
N H
N H
O m
O O
X
N H
O
O
N H O
H N
O N N
N N
NH2
Figure 6. Synthesis of folate terminated polyrotaxanes. Calicetti et al [47, 48] synthesized the CD-FA conjugates, in which b-CD was connected to FA through a polyethylene glycol (PEG) spacer of different length (Figure 7).
8
NH2-PEG-NH2
b-CD
b-CD
+ NH2
TsO
PEG
N H
O O O
H N
N
HN N
H2N
N O
O
TEA
N H
N
O
OH O
b-CD
O O N
HN H2N
N
N
H N
NH O N H
PEG
N H
OH O
Figure 7. Synthetic route to CD-FA conjugate linked by PEG spacers of various length. b-CD-PEG-FA possess properties inherent to all three of its components: b-CD provides the ability to encapsulate organic molecules to improve their water solubility and stability; PEG linker introduces flexibility into the structure, while folic acid assures targeting of FR overexpressing cancer cells. MD simulations reveal that PEG chain, due to its flexibility, folds up and directs FA carboxylic group into the cyclodextrin cavity, which may result in lower affinities towards various drugs than this of parent, unsubstituted CD, as shown for b-estradiol (Table 1). b-Estradiol forms complexes of 1:1 and 2:1 host : guest molar ratio. Described in detail MD simulations for 2:1 complex reveal that b-estradiol molecule is located in between two b-CD molecules without entering the macrocyclic cavity as it is partly occupied by FA. In the same paper the complexation of CD-PEG-FA with chlorambucil, a chemotherapeutic drug (used in treatment of chronic lymphocytic leukemia, Hodgkin lymphoma, and non-Hodgkin lymphoma) is described. 1:1 Complexes are formed, in which chlorambucil phenolic moiety is included inside the CD cavity, with chloroaliphatic chain dangling from CDs wider rim and carboxylic groups interacting with lower rim primary hydroxyl groups.
9
Figure 8. Synthetic route to b-CD-PEG-N3-FA conjugate. 5-Fluorouracil was used as a model drug by Zhang [49] to study the newly developed drug carrier built of b-CD, FA and poly(ethylene glycol) spacer of various length using the “click” strategy (b-CD-PEG-N3-FA, Figure 8).
10
In aqueous solution, the 1:1 supramolecular complex of 5-fluorouracil and a drug carrier conjugate formed nanoparticles, properties of which were evaluated against two cell lines (HeLa and A545) differing in the quantity of folate receptor. HeLa cells were affected in a greater extent which may imply that endocytosis mediated by folate receptors influences cellular uptake efficiency of the studied nanoparticles. Another b-CD-PEG-FA, where five hydroxyls were substituted with -PEG–NH2 group (b-CD-PEG5-FA) and one of them connected to folate, was used for a complexation of b-estradiol and chlorambucil [50].
3.1.3. Other cyclodextrin - folic acid conjugates. Okamatsu et al [51] developed another folate appended b-CD (b-CD-CA2-FA) for the delivery of DOX. In this new carrier, all b-CD primary hydroxyl groups were substituted by FA, separated by two caproic acid (CA) spacers (for synthesis see Figure 9). Such derivatized b-CD formed strong host : guest complex with DOX (>10 6 M-1) as compared to 102 for unsubstituted b-CD.
Figure 9. Synthetic route to b-CD-CA2-FA.
11
Figure 10. b-CD-s-Va-FA.
b-CD-CA2-FA increased the antitumor activity of DOX in FR-a positive cells (KB) but not in FR-a negative (A549) cells. The similar carriers, but based on all three native (a-, b-, and g-) cyclodextrins and possessing only one caproic acid spacer (CD-CA1-FA), were developed by the same group [52] and tested as potential drug delivery system for DOX. The stability of formed
1:1
inclusion
complexes
increases
in
the
series:
a-CD-CA1-FA/DOX
<
g-CD-CA1-FA/DOX < b-CD-CA1-FA/DOX. Out of the three new compounds, only derivative of the medium-sized b-CD increased the antitumor activity of doxorubicin and paclitaxel. Arima et al applied folate appended methylated b-CD (Me-b-CD-FA) for the complexation of DOX. The stability constant of the complex was much higher than this for the unsubstituted b-CD (3.0 × 105 M−1) [53]. Bilensoy et al [54] designed and introduced two new amphiphilic b-cyclodextrins for chemotherapeutic drug paclitaxel (PCX), diterpenoid obtained from a Pacific yew tree, exhibiting anticancer activities in breast cancer, ovarian cancer, and non-small lung cancer [55]. The CD used, was modified in the secondary face with hexanoyl chain, ending with folate and with remaining 13 secondary hydroxyl groups protected by valerate (b-CD-s-Va-FA,
12
Figure 10). Analogous cyclodextrin modified at the primary face (b-CD-p-Va-FA) was also obtained. Both of the synthesized CDs formed nanoparticles with PCX, where the drug molecule was placed in the space between the hydrophobic chains. Blank nanoparticles showed indiscernible cytotoxicity, while PCX-FA-CD nanoparticles exhibited high anticancer efficacy as a result of the increased interaction with the FA receptor cancer cells. Liu et al [56] prepared g-CD-FA conjugate as a drug delivery system for an enhancement of the anticancer effect of carboplatin (CBP). CBP was conjugated to C 60 and subsequently encapsulated in the cavity of g-CD host. C60-g-CD-FA improved the intracellular uptake and as well as a release of carboplatin which resulted in the higher cytotoxicity effect against HeLa cells. Su and Zhao [57] developed nanoparticles based on carboxymethyl-b-CD (CO-Me-b-CD-FA) conjugated bovine serum, decorated with folate and loaded them with 5-FU. They proved, that the newly synthesized NPs have an influence on the folate receptor mediated endocytosis and induce higher intracellular uptake of 5-FU leading to increased apoptosis as compared to the free drug. Zhou et al [58] developed biodegradable polymer micelles built of b-CD and pluronic F127-bpoly(e-caprolactone) copolymer (CPL) decorated with folate (b-CD-CPL-FA). Such prepared micelles were loaded with DOX and its release in vitro and in vivo at different temperatures and pH was studied. Cellular uptake studies (using HepG2, KB and A549 cell lines) showed that DOX loaded micelles inhibit significantly tumor growth. They, however, do not target normal fibroblast cells, which is due to the specific folate targeting.
13
Figure 11. Schematic synthesis of polymer micelles built of b-CD and pluronic F127-b-poly(ecaprolactone) copolymer (CPL) decorated with folate (b-CD-CPL-FA).
14
Ozeki et al [59] proposed a different approach to application of folate-based CDs. They developed another b-CD-FA-DOX conjugate (DOX7-ss-b-CD-FA7, Figure 12), where all O-3 primary hydroxyl groups in b-CD were substituted with DOX connected by disulfide bond. Disulfide bond ensures the dissociation of the conjugate under acidic conditions (like in endosomes [60]) and release of DOX. The obtained b-CD-DOX exhibited increased cellular uptake as compared to the free doxorubicin in EMT6/P cells and, more importantly, had a significant cytotoxic effect in DOX-resistant EMT6/AR1 cells.
Figure 12. Synthetic route towards b-CD-(FA)7-SS-DOX. Table 1. Main features describing the host : guest complexes of folate appended CDs with various drugs. 15
Host
Guest
Method of
Stoichio
Methods used
Association
Ka (M-1)
complex
metry
for the
constant
with
preparation
(H:G)
description of
-1
Ka (M )
complexes EN-b-CD-F
MTX
paste
1:1
NMR
ted CD a*
3,89*102 [61]
A EN-b-CD-F
unsubstitu
SCU
Solvent
1:1
evaporation
A
NMR, solubility
3.6 *102
6,34*102
10.9*102
6,34*102
studies XRD, TGA, SEM
DETA-b-CD
SCU
Solvent
1:1
evaporation
-FA
NMR, solubility studies XRD,
[62]
TGA, SEM TETA-b-CD
SCU
Solvent
1:1
evaporation
-FA
1.49*103
6,34*102
Molecular
1.53*104 (for
7,79*104
dynamics,
1:1)
NMR, solubility studies XRD, TGA, SEM
b-CD-PEG-
b-EST
Co-
2:1, 1:1
solubilization
FA
solubility studies b-CD-PEG-
CMBL
FA b-CD-PEG-
5-FU
FA b-CD-CA2-
DOX
Co-
1:1
solubilization
dynamics
Freeze
UV-VIS ,
drying
NMR
Co-
1:1
solubilization
FA
Molecular
Surface
a*
a*
a*
a*
2.4*106
2,2*102
plasmon resonance, Fluorescence
a-CD-CA1-
DOX
DOX
A
Fluorescence
2.1*104
2,0*102 [63]
Co-
1:1
Fluorescence
1.7*106
2,2*102
1:1
Fluorescence
5.3*104
2,2*102
solubilization
FA g-CD-CA1-F
1:1
solubilization
FA b-CD-CA1-
Co-
DOX
Cosolubilization
16
Me-b-CD-FA
DOX
-
1:1
-
3.0 × 105
2,2*102
a* not provided
3.2. Dendrimers.
Dendrimers are highly branched, spherical polymers with a symmetrical core, an inner shell, and an outer shell. Their biomedical applications include tissue engineering, drug delivery, and gene transfer[64]. There are numerous examples of various polymers appended with cyclodextrins applied for gene delivery [65]. Arima et al [66] developed polyethylene glycol (PEG) and folate-PEG derived CDs, as well as polypseudorotaxane-appended CDs for sustained release of DNA and siRNA. The same authors [67, 68] reported the synthesis of a G3 generation polyamidamine (PANAAM) dendrimers based on a-CD for siRNA transfer in the binary and ternary systems (siRNA/carrier; pDNA/siRNA/carrier, accordingly). Subsequently, to enhance both siRNA delivery as well as target specificity of the developed carrier, the substitution of a-CD (G3) with various amounts of folate was performed. Such obtained carriers were tested for their siRNA delivery properties towards folate receptor CCs both in vitro and in vivo [69]. a-CD (G3)-FA-siRNA complex showed cytotoxicity neither in FR-positive KB cells nor in FR-negative A549 cells. It exhibited RNA interference effects (RNAi) in luciferase assay system. Bearing in mind all these facts, the authors suggested that the newly developed a-CD (G3)-FA conjugate may be used as a siRNA carrier for a folate receptor overexpressing cancer cells. Nevertheless a-CD (G3)-FA did not show any significant effect on the RNA interference in vivo (experiments carried out with mice bearing colon-26 cancer cells). The easiest way to enhance the siRNA transfer activity of the dendrimer is to increase its generation. This may, however, cause its higher toxicity [70]. Arima et al [71] continued their work on a-CD–FA dendrimers developing a new class of generation 4 (G4) dendrimers (a-CD (G4)-FA, Figure 13), which - due to the incorporation of the PEG chains in their structure for the formation of hydration layer - resulted in a high safety profile and improved a blood circulating ability.
17
Figure 13. Schematic structure of G4 a-CD–FA dendrimer.
They were tested as carriers for tumor targeting siRNA both in vitro and in vivo and exhibited superior to G3 dendrimers RNAi effects. Moreover, the authors imply the possibility of application of G3 a-CD–FA dendrimers as a folate receptor overexpressing cancer cell selective DNA carriers. Qiu et al [72] developed a series of self-assembling micelles based on star-shaped b-CD-caprolactone amphiphilic copolymers and subsequently, micellar carriers based on a folate conjugated CD-caprolactone polymer (FA-CD-PEL). In vitro and in vivo studies of FA-CD-PEL proved that they enhance the antitumor efficacy of doxorubicin while reducing its toxicity in non-cancer cells [73].
Table 2. A summary of in vitro and in vivo studies of drug carriers based on folateappended cyclodextrins. Cyclodextrin
EN-b-CD-FA [42]
Drug MTX
Cancer type In vitro - Breast (MCF-7) - Lung (A549)
EN-b-CD-FA [44]
DCX
In vitro -
Cervical (HeLa)
-
Oral (KB)
-
Liver (HepG2)
18
Observed effects - Lower (four times) cytotoxity towards normal cell lines and higher cytotoxity towards studied cancer lines (as compared to free MTX). Cytotoxity comparable to free DTX. Vialability of the cells decreases in the series OB > HepG2> HeLa > KB -
In vitro
b-CD-CA2-FA [51]
-
Oral (KB)
-
Lung (A549)
In vivo - Colon (mice inoculated with Colon26, cells, an FR-αpositive mouse colon carcinoma cell line)
a-CD-CA1-FA [52]
DOX
In vitro -
b-CD-CA1-FA [52]
DOX
In vitro -
g-CD-CA1-FA [52]
DOX
DOX
Oral (KB)
In vivo - Colon (mice inoculated with Colon26, cells, an FR-αpositive mouse colon carcinoma cell line) In vitro -
Me-b-CD-A [53]
Oral (KB)
Oral (KB)
In vitro -
Oral (KB)
19
cells expressing higher FR levels are more sensitive to formulations containing folate. Increase of DOX antitumor activity in KB Increase of DOX cellular uptake (2x) in KB Inhibition of DOX cellular efflux (KB) No change in antitumor activity in A549
Inhibition of tumor growth, as compared to control and DOX alone after intratumoral administration Inhibition of tumor growth after intravenous injection of the complex (contrary to lack of inhibition in case of free DOX injection) No change in anticancer activity as compared to free DOX
Increase of DOX cellular uptake Inhibition of DOX cellular efflux Increased antitumor activity, as compared to free DOX, after intratumoral and intravenous administration to tumor-bearing mice No change in anticancer activity as compared to free DOX Increased antitumor activity, as compared to free DOX Increase of DOX
cellular uptake No effect on DOX cellular efflux
In vivo Colon (mice inoculated with Colon-26, cells, an FR-α-positive mouse colon carcinoma cell line)
b-CD-s-Va-FA [54]
PCX
In vitro Breast cancer (T47D and ZR-75-1)
b-CD-p-Va-FA [54]
PCX
In vitro Breast cancer (T47D and ZR-75-1)
C60-g-CD-FA [56]
CBP
In vitro Cervical (HeLa)
b-CD-CPL-FA [58]
DOX7-ss-b-CD-FA7 [59]
DOX
DOX
In vitro -
Liver (HepG2)
-
Oral (KB)
-
Lung (A549)
In vitro -
Breast (mouse EMT6/P)
-
Breast (mouse, EMT6/AR1 - resistant to DOX)
-
Lung (A549)
20
Inhibition of tumor growth after intravenous injection of the complex Significant improvement of the mice survival rate, as compared to control (5% mannitol), free DOX and DOX/M-b-CD complex On T-47D cellscomparative anticancer efficacy to that of free PCX On ZR-75-1 cells significant improvement of anticancer efficacy on cells On T-47D cellscomparative anticancer efficacy to that of free PCX On ZR-75-1 cells significant improvement of anticancer efficacy on cells Increased antitumor activity, as compared to free CBP Increase of DOX cellular uptake in HepG2 and KB
Increased cellular uptake in EMT6/P and EMT6/AR1 cells Cellular uptake in A549 similar to that of free DOX Reduced efflux of DOX from EMT6/P and EMT6/AR1 cells No change in DOX efflux from A549 as
a-CD (G4)-FA [71]
siRNA
In vitro Oral (KB)
-
compared with that of free DOX - Antitumor activity was observed
In vivo -
Colon (mice inoculated with Colon26, cells, an FR-αpositive mouse colon carcinoma cell line)
-
-
FA-CD-PEL [73]
DOX
In vitro -
Cervical (HeLa)
-
Oral (KB)
-
Lung (A549)
In vivo - Oral (KB cellxenografted nude mouse model)
Inhibition of tumor growth No severe side effects under the experimental conditions Significant increase of the blood half-life of si RNA within the complex
Increased cellular uptake in HeLa cells and to the lesser extent in KB cells No change in cellular uptake in A549 cells - Significant improvement of the mice survival rate
4. Folate receptor targeting via other delivery agents. Comparison with folate appended cyclodextrins.
Apart from folate-based cyclodextrins there is a great number of other folate-based carriers described in the literature, including simple folate as well as liposome-folate conjugates and folate-decorated nanoparticles. All of the discussed carriers seem to be superior to the free drug due to the presence of the FA moiety which ensures FR targeting. On the other hand, folate moiety in the carrier competes with free the FA for the same receptor binding sites. The detailed analysis of advantages and disadvantages of specific drug delivery systems is presented in Table 2. Drug-folate conjugates are
21
designed by a covalently linking the drug molecule to a folate carrier through an easy to cleave spacer, which allows the drug to be released at the cellular target site. This approach, is usually time and money consuming. There is a large number of studies concerning delivery of chemotherapeutic agents with the application of liposomes. In 1998 Lee and Low reported the synthesis of folate-conjugated liposomes [74]. Later a number
of
data
concerning
such
derivatives,
including
folate-PEG-
distearoylphosphatidyletha-nolamine
[75],
folate-PEG-cholesterol,
folate-PEG-
cholesteryl [76], and folateglutathione-PEG-distearoyl-phosphatidylethanolamine [77], which improved FR targeted delivery of liposomes, were reported. Unfortunately, the folate moiety self-aggregates on the surface of the liposomes, which leads to limited FR targeting efficiency [78]. Many studies were dedicated to the application of folatedecorated nanoparticles [79, 80]. As with liposomes, PEG is often incorporated in their structure, in order to improve the solubility of the carrier and subsequently solubility and half-life of the drug. FA linked to nanoparticles ensures an extended circulation time and guides nanoparticle-based carriers to FR-positive tumors [81]. The huge drawback of such approach is an accumulation of FA-decorated nanoparticles in the liver and kidneys, which may result in damaging their proper functioning [82]. As FA-decorated nanoparticles compete with free FA for the same receptor binding sites, the development of nanoparticles which can bind more effectively to folate receptor is also a major issue. Addition of more FA groups on the nanopoparticle surface seems to be favorable; nevertheless there is an optimal amount of FA, above which the uptake of FAbased-nanocarriers decreases [83]. In general, inclusion complexes of drugs with CDs are characterized by better solubility in water and better drug bioavailability. Nevertheless, the use of only very stable host: guest complexes (Ka values of at least 104−105 M−1) may find a practical application, as weaker complexes rapidly dissociate into free CDs and free drugs after parenteral administration [84,85]. To conclude, all of the discussed carriers improve the activity of the drug, nevertheless application of each class of these molecules has some drawbacks, that still need to be addressed.
22
Table 3. Comparison of various folate-based carriers. Drug-delivery system
Advantages
Disadvantages
Drug-folate conjugate
Specific FR targeting Minimization of systemic distribution of the drug
The amount of a drug attached to the folate carrier (drug loading) is limited due to the available number of attachment sites. Development of the new conjugates and their subsequent synthesis is often time-consuming and expensive - Competition with free FA for the same receptor binding sites
Liposome-folate-encapsulated
Specific FR targeting Ability for encapsulation and delivery of a large quantity of a drug in a single carrier More efficient transport of the drug molecule leading to its better uptake and higher cytoxicity
Selfaggregation of folate on the surface of liposomes limits their potential application Liposome stabilizing factors, such as incorporating PEG chains into the structure, prevent efficient unloading of the drug which follows uptake by a target cell - Competition with free FA for the same receptor binding sites
Specific FR targeting Ability for encapsulation and delivery of a large quantity of a drug in a single carrier
- Significant amounts of FA-decorated nanoparticles accumulate in the liver and kidneys. Accumulation of FAdecorated nanoparticles containing chemotherapeutic drugs in the kidneys increases the risk of impairing their proper functioning - FA-decorated nanoparticles compete with free FA for the same receptor binding sites - Manufacturing issues (scalability and batch-to-batch consistency)
drug
Folate-decorated nanoparticles
Folate appended
-
Specific FR
23
-
The amount of
cyclodextrin/drug inclusion complex
targeting Increase of the water solubility of hydrophobic drugs Increase of the drug’s bioavailability
a drug inside a single carrier is limited due to the limited inclusion possibility (based on the complex stoichiometry) Competition with free FA for the same receptor binding sites - In vivo application: only complexes of Kas>104 M-1 do not dissolve
5. Conclusions. The application of monosubstituted cyclodextrins, CDs with a higher degree of substitution, as well as various polymers derivatized with folate, as targeted drug delivery systems was presented. The majority of conjugates are based on medium-sized b-CD, as it usually forms more stable complexes with studied guest compounds than other two native (a- and g-) CDs. Drugs such as methotrexate, b-estradiol, chlorambucil, scutellarin, 5-fluorouracil, doxorubicin, paclitaxel, and carboplatin, in most cases formed 1:1 inclusion complexes. The comparison of the stability of the drug/FA-appended cyclodextrin complexes with the corresponding complexes based on a parent unsubstituted cyclodextrin proved that, in most cases, FA moiety covalently linked to the cyclodextrin core improves the stability of the complex. It is most pronounced for doxorubicin complexes (Ka three orders of magnitude higher for Me-b-CD-FA and four orders of magnitude higher for b-CD-CA1-FA and b-CD-CA2-FA than for native b-CD). Synthetic procedures leading to FA-CD conjugates were presented in detail. The majority of the complexes were studied only in vitro, with only a number studied also in vivo. In vitro studies were usually performed in FR-positive cells and often compared with FR-negative cells. The results obtained from these studies suggest the superior properties of the studied systems as compared to the free drug, mainly in the improved anticancer activity, increased drug cellular uptake, and limited drug cellular efflux. Folate appended cyclodextrins were also found to be good sustained release systems for DNA and siRNA. Comparison of the folate-appended cyclodextrins with other FA-based drug24
carriers including simple folate, liposome-folate conjugates, and folate-decorated nanoparticles was also performed. All of the discussed carriers improve the activity of the drug; nevertheless an application of each class of these molecules has several drawbacks, that still need to be addressed. Definitely, the application of cyclodextrins avoids the risk of disadvantages characteristic to other carriers, such as accumulation of FA-decorated nanoparticles containing chemotherapeutic drugs in kidneys leading to impairment of their proper functioning, or manufacturing issues (scalability and batch-tobatch consistency). Nevertheless, the necessity of the application of only very strong complexes (of Ka values of at least 104−105 M−1) to maintain their non-dissociated form in vivo after parenteral administration, creates a great challenge for the design and efficient synthesis of proper CD-based carriers. At the moment the clinical applications of folateappended cyclodextrins for drug, DNA and siRNA are still at prospects of the future. Further investigations are required regarding pharmacokinetics, and safety profiles, along
with
detailed
understanding
of
mechanisms
by which
folate-appended
cyclodextrins enhance antitumor activity of chemoterapeutic drugs. Nevertheless, these new carriers make a very promising class of FR-a targeting delivery systems.
Acknowledgements This work was financed by the Polish Ministry of Science and Higher Education, Grant No. IP2012007472. References: [1] C.S. Kue, A. Kamkaew, K. Burgess, L.V. Kiew, L.Y. Chung, H.B. Lee, Med. Res. Rev. 2016, 494. [2] Y. Zhong, F. Meng, C. Deng, Z. Zhong, Biomacromol, 2014, 15, 1955. [3] A.M. Gazzali, M. Lobry, L. Colombeau, S. Acherar, H. Azaïs, S. Mordon, P. Arnoux, F. Baros, R. Vanderesse, C. Frochot, Eur. J. Pharm. Sci. 2016, 93, 419. [4] C. Chen, J. Ke, X.E. Zhou, W. Yi, J.S. Brunzelle, J. Li, E-L. Yong, H.E. Xu, K. Melcher, Nature, 2013, 500,486. [5] X. Guo, C.L. Shi, J. Wang, S.B. Di, S.B. Zhou, Biomater., 2013, 34, 4554. 25
[6] B. Sahoo, K.S.P. Devi, R. Banerjee, T.K. Maiti, P. Pramanik, D. Dhara, ACS Appl. Mater. Interfaces, 2013, 5, 3884. [7] Chaudhury, S. Das, Curr. Pharm. Biotechnol. 2015, 16, 333. [8] S. Santra, C. Kaittanis, O.J. Santiesteban, J.M. Perez, J. Am. Chem. Soc., 2011, 133, 16680. [9] J.W. Lee, J.Y. Lu, P.S. Low, P.L. Fuchs, Bioorg. Med. Chem., 2002, 10, 2397. [10] W.A. Henne, D.D. Doorneweerd, A.R. Hilgenbrink, S.A. Kularatne, P.S. Low, Bioorg. Chem. Lett., 2006, 16, 5350. [11] I.R. Vlahov, C.P. Leamon, Bioconjugate Chem., 2012, 23,1357. [12] A. Guaragna, A. Chiaviello, C. Paolella, D. D’Alonzo, G. Palumbo, G. Palumbo, Bioconjugate Chem., 2012, 23, 84. [13] Y. Singh, M. Palombo, P.J. Sinko, Curr. Med. Chem., 2008, 15, 1802. [14] J. Szejtli, Chem. Rev., 1998, 98, 1743. [15] M.E. Brewster, T. Loftsson, Adv. Drug Deliv. Rev., 2010, 59, 645. [16] M. Ceborska, Curr. Org. Chem., 2014, 18, 1878. [17] H-J. Schmannan, E. Schollmeyer, J. Cosmet. Sci., 2002, 53, 185. [18] G. Astray, C. Gonzalez-Barreiro, J.C. Mejuto, R. Rial-Otero, J. Simal-Gandara, Food Hydrocolloid., 2009, 23, 1631. [19] J. Yin, Z.W. Zhou, S.F. Zhou, Drug Deliv. Transl. Res., 2013, 3, 364. [20] T. Irie, E. Uekama, J. Pharm. Sci., 1997, 86, 147. [21] T. Loftsson, M.E. Brewster, J. Pharm. Pharmacol., 2010, 62, 1607. [22] J. Zhang, P.X. Ma, Adv. Drug Deliv. Rev. 2013, 65, 1215. [23] F.M. Sirotnak, B.Tolner, Annu. Rev. Nutr., 1999, 19, 91. [24] D.W. Horne, K.A. Reed, J. Hoefs, H.M. Said, Am. J. Clin. Nutr., 1993, 58, 80. [25] Y.G. Assaraf, C.P. Leamon, J.A. Reddy, Drug Resist. Updat., 2014, 17, 89. [26] J. Shen, K.S. Putt, D.W. Visscher, L. Murphy, C. Cohen, S. Singhal, G. Sandusky, Y. Feng, D.S. Dimitrov, P.S., Oncotarget, 2015, 6, 14700. [27] S.D. Weitman, A.G. Weinberg, L.R. Coney, V.R. Zurawski, D.S. Jennings, B.A. Kamen, Cancer Res., 1992, 52, 6708.
26
[28] D.J. O’Shannessy, E.B. Somers, E. Albone, X. Cheng, Y.C. Park, B.E. Tomkowicz, Y. Hamuro, T.O. Kohl, T.M. Forsyth, R. Smale, Y-S. Fu, N.C.Nicolaides, Oncotarget. 2011, 2, 1227. [29] H. Kurahara, S. Takao, T. Kuwahata, T. Nagai, Q. Ding, K. Maeda, H. Shinchi, Y. Mataki, K. Maemura, T. Matsuyama, S. Natsugoe, Ann. Surg. Oncol. 2012, 19, 2264. [30] J. Shen, A.R. Hilgenbrink, W. Xia, Y. Feng, D.S. Dimitrov, M.B. Lockwood, R.J. Amato, P.S. Low, J. Leukoc. Biol., 2014, 96, 563. [31] Y. Tian, G. Wu, J.C. Xing, J. Tang, Y. Zhang, Z.M. Huang, Z.C. Jia, R. Zhao, Z.Q. Tian, S.F. Wang, X.L. Chen, L. Wang, Y.Z. Wu, B. Ni, BMC Immunol., 2012, 13, 30. [32] E.I. Sega, P.S. Low, Cancer Metastasis Rev., 2008, 27, 655. [33] L. Teng, J. Xie, L. Teng, R.J. Lee, Expert. Opin. Drug. Deliv., 2012, 9, 901. [34] H. Shi, J. Guo, C. Li, Z. Wang, Drug Des. Devel. Ther., 2015, 9, 4989. [35] K. Chmurski, P. Stępniak, J. Jurczak, Carbohydr. Polym., 2016, 138, 8. [36] M. Ceborska, M. Zimnicka, M. Pietrzak, A. Troć, M. Koźbiał, J. Lipkowski, Org. Biomol. Chem., 2012, 10, 5186. [37] M. Zimnicka, A. Troć, M. Ceborska, M. Jakubczak, M. Koliński, W. Danikiewicz, Anal. Chem., 2014, 86, 4249. [38] M. Ceborska, M. Zimnicka, M. Wszelaka-Rylik, A Troć, J. Mol. Struc., 2016, 1109, 114. [39] A. Clementi, M.C. Aversa, C. Corsaro, J. Spooren, R. Stancanelli, C O’Connor, M. McNamara, A. Mazzaglia, J. Incl. Phenom. Macrocycl. Chem., 2011, 69, 321. [40] Z. Tofzikovskaya, C. O’Connor, M. McNamara, J. Incl. Phenom. Macrocycl. Chem., 2012, 74, 437. [41] Z. Tofzikovskaya, A. Casey, O. Howe, C. O’Connor, M. McNamara, J. Incl. Phenom. Macrocycl. Chem., 2015, 81, 85. [42] B. Yang, L. Zhao, X. Yang, X.L. Liao, J. Jang, J.H. Zhang, C.Z. Gao, Carbohydr. Polym., 2013, 92, 1308. [43] R. Liao, Y. Zhao, X. Liao, M. Liu, C. Gao, J. Yang, B. Yang, Polym. Adv. Technol., 2015, 26, 487. [44] J. Xu, B. Xu, D. Shou, F. Qin, Y. Xu, Y. Hu, Eur. J. Pharm. Sci., 2016, 83, 132. 27
[45] H. Yao, S.S. Ng, W. O. Tucker, Y-K-T. Tsang, K. Man, X-M. Wang, B.K.C. Chow, HF. Kung, G-P. Tang, M.C. Lin, Biomater., 2009, 30, 5793. [46] Y. Zhou, H. Wang, C. Wang, Y. Li, W. Lu, S. Chen, J. Luo, Mol. Pharm., 2012, 9, 1067. [47] P. Calicetti, S. Salmaso, A. Semenzato, T. Carofiglio, R. Fornasier, M. Fermeglia, M. Ferrone, S. Pricl, Bioconjugate Chem., 2003, 14, 899. [48] S. Salmaso, A. Samenzato, P. Caliceti, J. Hoebeke, F. Sonvico, C. Dubernet, P. Couvreur, Bioconjugate Chem., 2004, 15, 997. [49] H. Zhang, Z. Cai, Y. Sun, F. Yu, Y. Chen, B. Sun, J. Biomed. Res. Mater. Part A, 2012, 100A, 2441. [50] S. Salamaso, S. Bersani, A. Semenzato, P. Caliceti, J. Drug Target, 2007, 15, 379. [51] A. Okamatsu, K. Motoyama, R. Onodera, T. Higashi, T. Koshigoe, Y. Shimada, K. Hattori, T. Takeuchi, H. Arima, Bioconjugate Chem., 2013, 24, 724. [52] A. Okamatsu, K. Motoyama, R. Onodera, T. Higashi, T. Koshigoe, Y. Shimada, K. Hattori, T. Takeuchi, H. Arima, Biomacromol., 2013, 14, 4420. [53] K. Motoyama, R. Onodera, A. Okamatsu, T. Higashi, R. Kariya, S. Okada, H. Arima, J. Drug. Target., 2014, 22, 211. [54] N. Erdogar, G. Esendagli, T.T. Nielsen, M. Sen, L. Oner, E. Bilensoy, Int. J. Pharm. 2016, 509, 375. [55] M. Agueros, V. Zabaleta, S. Espuelas, M.A. Campanero, J.M. Irache, J. Control. Release, 2010, 145, 2. [56] Y. Liu, Y. Pu, L. Sun, H. Yao, B. Zhao, R. Zhang, Y. Zhang, J. Biomed. Nanotechnol., 2016, 12, 1393. [57] C. Su, H. Li, Y. Shi, G. Wang, L. Liu, L. Zhao, R. Su, Int. J. Pharm., 2014, 474, 202. [58] Q. Zhou, X. Guo, T. Chen, Z. Zhang, S. Shao, C. Luo, J. Li, S. Zhou, J. Phys. Chem. B, 2011, 115, 12662. [59] H. Mizusako, T. Tagami, K. Hattori, T. Ozeki, J. Pharm. Sci., 2015, 104, 2934. [60] J. Yang, H. Chen, I.R. Vlahov, J.X. Cheng, P.S. Low, Proc. Natl. Acad. Sci. U.S.A., 2006, 103, 13872. [61] F. Pattarino, L. Giovannelli, G.B. Giovenzana, M. Rinaldi, M. Trotta, J. Drug Deliv. Sci. Technol., 2005, 15, 465. 28
[62] B. Yang, L.-J. Yang, J. Lin,Y. Chen,Y. Liu, J. Incl. Phenom. Macrocycl. Chem., 2009, 64, 149. [63] R. Anand, S. Ottani, F. Manoli, I. Manet, S. Monti, RSC Adv.,2012, 2, 2346. [64] M.A. Mintzer, M.W. Grinstaff, Chem. Soc. Rev., 2011, 40, 173. [65] H. Arima, K. Motoyama, T. Higashi, Adv. Drug Deliv. Rev., 2013, 65, 1204. [66] H. Arima, K. Motoyama, T. Higashi, Curr. Top. Med. Chem., 2014, 14, 465. [67] T. Tsutsumi, F. Hirayama, K. Uekama, H. Arima, J. Controlled Release, 2007, 119, 349. [68] H. Arima, T. Tsutsumi, A. Yosshimatsu, H. Ikeda, K. Motoyama, T. Higashi, F. Hirayama, K. Uekama, Eur. J. Pharm. Sci., 2011, 44, 375. [69] H. Arima, A. Yoshimatsu, H. Ikeda, A. Ohyama, K. Motoyama, T. Higashi, A. Tsuchiya, T. Niidome, Y. Katayama, K. Hattori, T. Takeuchi, Mol. Pharm., 2012, 9, 2591. [70] F. Kukowska-Latallo, A.U. Bielinska, J. Johnson, R. Spindler, D.A. Tomalia, J.R. Baker, Jr., Proc. Natl. Acad. Sci. U.S.A., 1996, 93, 4897. [71] A. Ohyama, T. Higashi, K. Motoyama, H. Arima, Bioconjugate Chem., 2016, 27, 521. [72] L.Y. Qiu, R.J. Wang, C. Zheng, Y. Jin, Nanomedicine, 2010, 5, 193. [73] L. Zhang, J. Lu, Y. Jin, L. Qiu, Colloids Surf. B Biointerfaces, 2014, 122, 260. [74] R.J. Lee, P.S. Low, BBA. Biomembranes, 1995, 1233, 134. [75] A. Gabizon, A.T. Horowitz, D. Goren, D. Tzemach, F. Mandelbaum-Shavit, M.M. Qazen, S. Zalipsky, Bioconjugate Chem. 1999, 10, 289. [76] W. Guo, T. Lee, J. Sudimack, R.J. Lee, Liposome Res., 2000, 10,179. [77] G. Xiang, J. Wu, Y. Lu, Z. Liu, R.J. Lee, Int. J. Pharmaceut., 2008, 356, 29. [78] Y. Liu, S. Xu2, L. Teng, B. Yung, J. Zhu, H. Ding, R.J. Lee, Anticancer Res., 2011, 31, 1521. [79] L. Brannon-Peppas, J.O. Blanchette, Adv. Drug Deliv. Rev., 2012, 64, 206. [80] L. Xu, Q. Bai, X. Zhang , H. Yang, J. Control. Release, 2017, 252, 73. [81] C. Yue, P. Liu, M. Zheng, P. Zhao, Y. Wang, Y. Ma, L. Cai, Biomater., 2013, 34, 6853. [82] J.S. Kim, M.H. Oh, J.Y. Park, T.G. Park, Y.S. Nam, Biomater., 2013, 34, 2370. 29
[83] N.V. Nukolova, H.S. Oberoi, S.M. Cohen, A.V. Kabanov, T.K. Bronich, Biomater., 2011, 32, 5417. [84] V.J. Stella, V.M. Rao, E.A. Zannou, V.V. Zia, Adv. Drug Deliv. Rev., 1999, 36, 3. [85] V.J. Stella, Q. He, Toxicol. Pathol., 2008, 36, 30.
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S0939-6411(17)30490-3 http://dx.doi.org/10.1016/j.ejpb.2017.09.005 EJPB 12590
To appear in:
European Journal of Pharmaceutics and Biopharmaceutics
Received Date: Revised Date: Accepted Date:
18 April 2017 1 September 2017 8 September 2017
Please cite this article as: M. Ceborska, Folate appended cyclodextrins for drug, DNA, and siRNA delivery, European Journal of Pharmaceutics and Biopharmaceutics (2017), doi: http://dx.doi.org/10.1016/j.ejpb. 2017.09.005
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Folate appended cyclodextrins for drug, DNA, and siRNA delivery Magdalena Ceborska*[a] ORCID 0000-0001-5555-771X
Institute of Physical Chemistry, Polish Academy of Sciences Kasprzaka 44/52, 01-224, Warsaw, Poland E-mail: [email protected]
Abstract Drug, DNA, and siRNA delivery systems based on cyclodextrin (CD) core and connected with folate (FA) via various linkers are presented. They include simple monoderivatized cyclodextrins as well as cyclodextrins with higher degree of substitution, both in their primary and secondary sides. Examples of simple polymers and dendrimers are also discussed. Such carriers possess properties inherent to both of their components. Cyclodextrin provides the ability to encapsulate organic molecules in its inner cavity, thus improving their solubility in water, bioavailability, and stability, while FA assures targeting folate receptor overexpressing cancer cells. Drug delivery systems loaded with drugs such as e.g. methotrexate, doxorubicin, paclitaxel, vinblastine, and docetaxel were found to have superior properties as compared to the free drug. Dendritic folate appended cyclodextrins were also found to be good sustained release systems for DNA and siRNA. The recent progress in the synthesis and drug, DNA, and siRNA delivery application of folate appended cyclodextrins is presented.
Keywords: cyclodextrin • folic acid • bioconjugate • drug delivery
1
1. Introduction Living cells depend upon vitamins for their proper function while cancer cells need them supplied in larger quantities for rapid growth sustenance. As vitamin uptake receptors (VUR) are often overexpressed on the surface of cancer cells, folate receptor (FR) is broadly utilized for tumor cells detection as well as for cancer-targeted drug delivery. There are two main strategies designed for targeted drug delivery to FR overexpressing tumor cells – first is a coupling of the desired drug to the monoclonal antibody against the FR, second a conjugation with folic acid (FA, Figure 1a) which shows high affinity to FR and thus good targeting properties [1,2]. FA (vitamin B9) is a small molecule built from p-aminobenzoate linked to a pteridine ring, and glutamic acid [3]. It possesses in its structure functionalities that allow for a FA conjugation to various molecules in order to form specific prodrugs. Conjugation via g-carboxyl group is preferred as it has no impact on the FA-receptor binding properties [4]. There is a great number of folate-based carriers described in the literature, including micelles [5], nanoparticles [6], liposome-folate-encapsulated drugs [7], and cytotoxic drugs-folate conjugates [8-13]. To improve the water solubility of drugs, encapsulation of bio-relevant molecules in cyclodextrins may be applied. To achieve both: tumor targeting properties and ability to increase water solubility, targeted drug delivery systems built from cyclodextrin covalently bonded with folate, are used. Native cyclodextrins (a-CD, b-CD, and g-CD) are macrocyclic oligosaccharides with a hydrophobic cavity and a hydrophilic external surface, which makes them readily soluble in water [14]. Their ability to form inclusion complexes with various organic molecules in solution [15] and in the solid state [16] is utilized by cosmetic [17] and food [18] industries. Derivatization of CDs leading to amphiphilic molecules increases their interactions with biological membranes, thus making them suitable for biomedical applications.[19] Their good water solubility combined with low immunogenicity [20] enables their broad application in pharmaceutical industry [21,22]. This paper features the recent progress in the synthesis and drug delivery application of folate appended cyclodextrins. Comparison of the folate-appended cyclodextrins with other FA targeted drug-carriers including simple folate, liposome-folate conjugates, and folatedecorated nanoparticles is also presented.
2
Figure 1.a) Folic acid b) native (a-, b-, and g-) cyclodextrins.
2. Mechanism of the folate mediated delivery of drugs to FR-a positive cancer cells. Folate is delivered into cells via the reduced folate carrier [23], the proton coupled folate transporter [24] or the folate receptor, which exists in four isoforms (FR-a, FR-b, FR-g, and FR-d) [25,26]. FR-a and FR-b are membrane-associated proteins anchored to a glycosylphosphatidylinositol (GPI) membrane. FR-a exhibits higher than FR-b affinity towards 5-methyltetrafolate. It is mainly expressed on the apical surfaces of epithelial cells [27,28], while FR-β is expressed on macrophages [29] and monocytes [30]. FR-γ was identified as a secretory protein from hematopoietic tissues and is characterized by much lower affinity towards folate than folate receptors FR-a and FR-b. The FR-d, which is hard to detect in human tissues, was only found on regulatory T cells [31]. The mostly studied FR-a is expressed at very low levels in normal tissues, but is overexpressed in many types of cancer. It is due to high demand of folate by rapidly dividing cells. Therefore, FR-a is utilized both as tumor cell marker, as well as the target for delivery of imaging and therapeutic agents [32,33]. The possible mechanism of targeted drug delivery via FA-CD conjugate/drug supramolecular complex is analogous to that of FA-drug covalent conjugate [34]. In a FA-drug conjugate drug molecule is linked to FA by easy to cleave linker (e.g. with disulfide bonds, Figure 2a), while in FA-CD conjugate/drug supramolecular complex it is kept in CD cavity by
3
weak interactions and hydrophobic forces (Figure 2b). The mechanism of the drug delivery to the FR-a positive cancer cells depends on the receptor-based endocytosis (Figure 2). The FA part of the drug delivery system ensures targeting of a folate receptor. Subsequently the FA-drug covalent conjugate (or FA-CD conjugate/drug supramolecular complex) enters endosome, where the drug is cleaved, which is followed by the free drug leaving the endosome to act upon cancer cells.
Figure 2. Mechanism of a folate-mediated delivery of drugs to FR-a positive cancer cells via receptor-mediated endocytosis a) using FA-drug conjugate b) using FA-CD/drug host: guest complex.
3. Folate-appended cyclodextrins as drug, DNA and siRNA delivery systems. The drug delivery systems based on cyclodextrins rely mainly on the formation of supramolecular host : guest inclusion complexes, where the bio-relevant molecule is wholly or partially encapsulated inside the macrocyclic cavity. While designing new potential receptors with cyclodextrin core, derivatized with various substituents, one has to bear in mind that covalently linked substituents may also penetrate the CD’s cavity thus hindering the inclusion of the guest molecule [35]. It is known that native (a-, b-, and g-) CDs are able to form associates with FA [36-38]. a-CD forms with FA weakly bound exclusion compound where guest molecule interacts with the secondary hydroxyls located at the wider rim of a-CD. band g-CDs form with FA pseudorotaxane structures, where the host molecule is threaded over
4
the guest FA. Therefore, to incorporate folate-appended CDs into the specific drug delivery systems, the requirement of the formation of stronger CD/drug than CD/FA complex should be fulfilled. In the present paper the recent progress in the synthesis and drug delivery application of folate appended cyclodextrins is presented. It can be clearly seen that b-CD is by far the most utilized cyclodextrin, with only a few reported examples of FA derivatization of a- and g-CDs. In Figure 3, drug molecules complexed with CD-FA conjugates described in this review are presented. The main features of the complexes (method of preparation, stoichiometry, and values of association constants) are presented in Table 1 presented at the end of chapter 3 (only for the complexes where such data was reported). Whenever the mode of complexation was described it is also noted in the main text. Moreover, Table 2 presented at the end of chapter 4 gives a simplified information about the results of application of specific FAappended-CD/drug complex for specific tumors.
5
Figure 3. Targeted drugs: methotrexate (MTX, 1), scutellarin (SCTL, 2) b-estradiol (b-EST, 3) chlorambucil (CLBL, 4), 5-fluorouracil (5-FU, 5), doxorubicin (DOX, 6), paclitaxel (PCX, 7), carboplatin (CBP, 8). 3. 1. Simple substituted cyclodextrins and polymers. 3.1.1. Cyclodextrin - folic acid - amido conjugates. Clementi et al [39] first synthesized the b-CD-FA conjugate, where FA was linked to 6-[2-aminoethyl)amino]-6-deoxy-b-CD (EN-b-CD-FA) via FA carboxyl groups (a and g) to form amido derivatives (Figure 4, only g- isomer is shown). The synthetic route, enabling formation of only desirable g-isomer, was later improved by Tofzikovskaya et al [40]. The proposed receptor increases significantly the photostability of FA, thus enabling its broader and easier applications, e.g. in food industry. Later, the cytotoxity of EN-b-CD-FA was evaluated [41] using MTT assay, MCF-7 (breast), BEAS-23B (normal lung), A5649 (lung cancer), and HeLa(cervical) cell lines. EN-b-CD-FA showed no cytotoxicity towards the studied cell lines rendering it safe for medical use.
Figure 4. Synthesis of CD-FA g-amido-conjugate. The 1:1 host : guest inclusion complex with antifolate cytotoxic drug methotrexate (MTX) was synthesized. It exhibited lower (four times) cytotoxity towards normal cell lines and higher cytotoxity towards all of studied cancer lines (as compared to free MTX). Another drug subjected to FA-appended CD was scutellarin, flavone used in the inhibition of platelet aggregation and for the improvement of blood circulation [42]. To increase its low water solubility as well as bioavailability it was reacted with three polyamine b-CD-FA conjugates:
ethylenediamine-b-CD-FA(EN-b-CD-FA),
6
diethylamine-b-CD-FA
(DETA-b-CD-FA) and triethylamine-b-CD-FA (TETA-b-CD-FA) [43] giving 1:1 inclusion complexes. In all three complexes scuttelarin enters CDs with cavity through its wider rim. Upon formation of host : guest complex the solubility of scutellarin increased significantly (approximately 1.2*102, 2.3*102 and 3.1*102, respectively). Xu et al [44] used the CD-FA conjugate in which b-CD is linked by a g-carboxyl group of folic acid with the amino group of 6-monodeoxy-6-(2-aminoethyl)-β-CD for the delivery of docetaxel (DCX) Figure 7), a drug used in treatment of breast, lung, and prostate cancers. 1:1 Complex of DCX with CD-FA conjugate exhibited significant antitumor efficacy (leading to cell apoptosis of folate receptor cancer cells). 3.1.2. Cyclodextrin - folic acid conjugates linked by polythylenimine and polyethylene glycol spacers of various lengths. Tang et al [45] developed a folate-polythylenimine-cyclodextrin (CD-PEI-FA) polymer which both in vitro and in vivo proved to be efficient gene delivery system which may be used in cancer gene therapy (Figure 5). Polyethylenimines (PEI) of various lengths are characterized by good transfection efficiency in the wide range of cells and are known to form polyplexes with DNA. The obtained FA-PEI-CD polymer allowed for a valuable transfection of broad array of cancer cells with low cytotoxicity.
b-CD 1)CDI b-CD 2) PEI-600, Et3N H N
N H
N H m1
FA
m2
CDI, NEt3N
b-CD
N H
H N
N H m1
O
O
H N m2
O O
N
n
O N H
O N H
HO O
Figure 5. Synthesis of CD- PEI-FA.
7
H N
O N N
NH N
O
O
H N
NH2
N H
O O
n
Folate terminated polyrotaxanes (FPP) consisted of a-CD decorated with PEI600 threaded over PEG chained capped with FA molecules at both sides were prepared (Figure 6) [46]. FPP/pDNA polyplexes were obtained and were shown to possess low cytotoxicity and high efficiency of DNA delivery to target cells.
a-CD
O H2N
O
O
NH2
m
H2N
O m
NH2
X
FA-NHS O O N H2N
H N
N N
O
O
N H
N H
N
O O m
O
X
O
N H
O
O
N H
O
H N
N N
N N
O
H2 N
N
CDI
H N
NH2
N m1 H
m2
O N
NH2
O
H N
N
N H2N
N
N
H N
H N
N
N H
N
N
N
NH2
NH2 O
O O
NH2
n O
O
N H
N H
O m
O O
X
N H
O
O
N H O
H N
O N N
N N
NH2
Figure 6. Synthesis of folate terminated polyrotaxanes. Calicetti et al [47, 48] synthesized the CD-FA conjugates, in which b-CD was connected to FA through a polyethylene glycol (PEG) spacer of different length (Figure 7).
8
NH2-PEG-NH2
b-CD
b-CD
+ NH2
TsO
PEG
N H
O O O
H N
N
HN N
H2N
N O
O
TEA
N H
N
O
OH O
b-CD
O O N
HN H2N
N
N
H N
NH O N H
PEG
N H
OH O
Figure 7. Synthetic route to CD-FA conjugate linked by PEG spacers of various length. b-CD-PEG-FA possess properties inherent to all three of its components: b-CD provides the ability to encapsulate organic molecules to improve their water solubility and stability; PEG linker introduces flexibility into the structure, while folic acid assures targeting of FR overexpressing cancer cells. MD simulations reveal that PEG chain, due to its flexibility, folds up and directs FA carboxylic group into the cyclodextrin cavity, which may result in lower affinities towards various drugs than this of parent, unsubstituted CD, as shown for b-estradiol (Table 1). b-Estradiol forms complexes of 1:1 and 2:1 host : guest molar ratio. Described in detail MD simulations for 2:1 complex reveal that b-estradiol molecule is located in between two b-CD molecules without entering the macrocyclic cavity as it is partly occupied by FA. In the same paper the complexation of CD-PEG-FA with chlorambucil, a chemotherapeutic drug (used in treatment of chronic lymphocytic leukemia, Hodgkin lymphoma, and non-Hodgkin lymphoma) is described. 1:1 Complexes are formed, in which chlorambucil phenolic moiety is included inside the CD cavity, with chloroaliphatic chain dangling from CDs wider rim and carboxylic groups interacting with lower rim primary hydroxyl groups.
9
Figure 8. Synthetic route to b-CD-PEG-N3-FA conjugate. 5-Fluorouracil was used as a model drug by Zhang [49] to study the newly developed drug carrier built of b-CD, FA and poly(ethylene glycol) spacer of various length using the “click” strategy (b-CD-PEG-N3-FA, Figure 8).
10
In aqueous solution, the 1:1 supramolecular complex of 5-fluorouracil and a drug carrier conjugate formed nanoparticles, properties of which were evaluated against two cell lines (HeLa and A545) differing in the quantity of folate receptor. HeLa cells were affected in a greater extent which may imply that endocytosis mediated by folate receptors influences cellular uptake efficiency of the studied nanoparticles. Another b-CD-PEG-FA, where five hydroxyls were substituted with -PEG–NH2 group (b-CD-PEG5-FA) and one of them connected to folate, was used for a complexation of b-estradiol and chlorambucil [50].
3.1.3. Other cyclodextrin - folic acid conjugates. Okamatsu et al [51] developed another folate appended b-CD (b-CD-CA2-FA) for the delivery of DOX. In this new carrier, all b-CD primary hydroxyl groups were substituted by FA, separated by two caproic acid (CA) spacers (for synthesis see Figure 9). Such derivatized b-CD formed strong host : guest complex with DOX (>10 6 M-1) as compared to 102 for unsubstituted b-CD.
Figure 9. Synthetic route to b-CD-CA2-FA.
11
Figure 10. b-CD-s-Va-FA.
b-CD-CA2-FA increased the antitumor activity of DOX in FR-a positive cells (KB) but not in FR-a negative (A549) cells. The similar carriers, but based on all three native (a-, b-, and g-) cyclodextrins and possessing only one caproic acid spacer (CD-CA1-FA), were developed by the same group [52] and tested as potential drug delivery system for DOX. The stability of formed
1:1
inclusion
complexes
increases
in
the
series:
a-CD-CA1-FA/DOX
<
g-CD-CA1-FA/DOX < b-CD-CA1-FA/DOX. Out of the three new compounds, only derivative of the medium-sized b-CD increased the antitumor activity of doxorubicin and paclitaxel. Arima et al applied folate appended methylated b-CD (Me-b-CD-FA) for the complexation of DOX. The stability constant of the complex was much higher than this for the unsubstituted b-CD (3.0 × 105 M−1) [53]. Bilensoy et al [54] designed and introduced two new amphiphilic b-cyclodextrins for chemotherapeutic drug paclitaxel (PCX), diterpenoid obtained from a Pacific yew tree, exhibiting anticancer activities in breast cancer, ovarian cancer, and non-small lung cancer [55]. The CD used, was modified in the secondary face with hexanoyl chain, ending with folate and with remaining 13 secondary hydroxyl groups protected by valerate (b-CD-s-Va-FA,
12
Figure 10). Analogous cyclodextrin modified at the primary face (b-CD-p-Va-FA) was also obtained. Both of the synthesized CDs formed nanoparticles with PCX, where the drug molecule was placed in the space between the hydrophobic chains. Blank nanoparticles showed indiscernible cytotoxicity, while PCX-FA-CD nanoparticles exhibited high anticancer efficacy as a result of the increased interaction with the FA receptor cancer cells. Liu et al [56] prepared g-CD-FA conjugate as a drug delivery system for an enhancement of the anticancer effect of carboplatin (CBP). CBP was conjugated to C 60 and subsequently encapsulated in the cavity of g-CD host. C60-g-CD-FA improved the intracellular uptake and as well as a release of carboplatin which resulted in the higher cytotoxicity effect against HeLa cells. Su and Zhao [57] developed nanoparticles based on carboxymethyl-b-CD (CO-Me-b-CD-FA) conjugated bovine serum, decorated with folate and loaded them with 5-FU. They proved, that the newly synthesized NPs have an influence on the folate receptor mediated endocytosis and induce higher intracellular uptake of 5-FU leading to increased apoptosis as compared to the free drug. Zhou et al [58] developed biodegradable polymer micelles built of b-CD and pluronic F127-bpoly(e-caprolactone) copolymer (CPL) decorated with folate (b-CD-CPL-FA). Such prepared micelles were loaded with DOX and its release in vitro and in vivo at different temperatures and pH was studied. Cellular uptake studies (using HepG2, KB and A549 cell lines) showed that DOX loaded micelles inhibit significantly tumor growth. They, however, do not target normal fibroblast cells, which is due to the specific folate targeting.
13
Figure 11. Schematic synthesis of polymer micelles built of b-CD and pluronic F127-b-poly(ecaprolactone) copolymer (CPL) decorated with folate (b-CD-CPL-FA).
14
Ozeki et al [59] proposed a different approach to application of folate-based CDs. They developed another b-CD-FA-DOX conjugate (DOX7-ss-b-CD-FA7, Figure 12), where all O-3 primary hydroxyl groups in b-CD were substituted with DOX connected by disulfide bond. Disulfide bond ensures the dissociation of the conjugate under acidic conditions (like in endosomes [60]) and release of DOX. The obtained b-CD-DOX exhibited increased cellular uptake as compared to the free doxorubicin in EMT6/P cells and, more importantly, had a significant cytotoxic effect in DOX-resistant EMT6/AR1 cells.
Figure 12. Synthetic route towards b-CD-(FA)7-SS-DOX. Table 1. Main features describing the host : guest complexes of folate appended CDs with various drugs. 15
Host
Guest
Method of
Stoichio
Methods used
Association
Ka (M-1)
complex
metry
for the
constant
with
preparation
(H:G)
description of
-1
Ka (M )
complexes EN-b-CD-F
MTX
paste
1:1
NMR
ted CD a*
3,89*102 [61]
A EN-b-CD-F
unsubstitu
SCU
Solvent
1:1
evaporation
A
NMR, solubility
3.6 *102
6,34*102
10.9*102
6,34*102
studies XRD, TGA, SEM
DETA-b-CD
SCU
Solvent
1:1
evaporation
-FA
NMR, solubility studies XRD,
[62]
TGA, SEM TETA-b-CD
SCU
Solvent
1:1
evaporation
-FA
1.49*103
6,34*102
Molecular
1.53*104 (for
7,79*104
dynamics,
1:1)
NMR, solubility studies XRD, TGA, SEM
b-CD-PEG-
b-EST
Co-
2:1, 1:1
solubilization
FA
solubility studies b-CD-PEG-
CMBL
FA b-CD-PEG-
5-FU
FA b-CD-CA2-
DOX
Co-
1:1
solubilization
dynamics
Freeze
UV-VIS ,
drying
NMR
Co-
1:1
solubilization
FA
Molecular
Surface
a*
a*
a*
a*
2.4*106
2,2*102
plasmon resonance, Fluorescence
a-CD-CA1-
DOX
DOX
A
Fluorescence
2.1*104
2,0*102 [63]
Co-
1:1
Fluorescence
1.7*106
2,2*102
1:1
Fluorescence
5.3*104
2,2*102
solubilization
FA g-CD-CA1-F
1:1
solubilization
FA b-CD-CA1-
Co-
DOX
Cosolubilization
16
Me-b-CD-FA
DOX
-
1:1
-
3.0 × 105
2,2*102
a* not provided
3.2. Dendrimers.
Dendrimers are highly branched, spherical polymers with a symmetrical core, an inner shell, and an outer shell. Their biomedical applications include tissue engineering, drug delivery, and gene transfer[64]. There are numerous examples of various polymers appended with cyclodextrins applied for gene delivery [65]. Arima et al [66] developed polyethylene glycol (PEG) and folate-PEG derived CDs, as well as polypseudorotaxane-appended CDs for sustained release of DNA and siRNA. The same authors [67, 68] reported the synthesis of a G3 generation polyamidamine (PANAAM) dendrimers based on a-CD for siRNA transfer in the binary and ternary systems (siRNA/carrier; pDNA/siRNA/carrier, accordingly). Subsequently, to enhance both siRNA delivery as well as target specificity of the developed carrier, the substitution of a-CD (G3) with various amounts of folate was performed. Such obtained carriers were tested for their siRNA delivery properties towards folate receptor CCs both in vitro and in vivo [69]. a-CD (G3)-FA-siRNA complex showed cytotoxicity neither in FR-positive KB cells nor in FR-negative A549 cells. It exhibited RNA interference effects (RNAi) in luciferase assay system. Bearing in mind all these facts, the authors suggested that the newly developed a-CD (G3)-FA conjugate may be used as a siRNA carrier for a folate receptor overexpressing cancer cells. Nevertheless a-CD (G3)-FA did not show any significant effect on the RNA interference in vivo (experiments carried out with mice bearing colon-26 cancer cells). The easiest way to enhance the siRNA transfer activity of the dendrimer is to increase its generation. This may, however, cause its higher toxicity [70]. Arima et al [71] continued their work on a-CD–FA dendrimers developing a new class of generation 4 (G4) dendrimers (a-CD (G4)-FA, Figure 13), which - due to the incorporation of the PEG chains in their structure for the formation of hydration layer - resulted in a high safety profile and improved a blood circulating ability.
17
Figure 13. Schematic structure of G4 a-CD–FA dendrimer.
They were tested as carriers for tumor targeting siRNA both in vitro and in vivo and exhibited superior to G3 dendrimers RNAi effects. Moreover, the authors imply the possibility of application of G3 a-CD–FA dendrimers as a folate receptor overexpressing cancer cell selective DNA carriers. Qiu et al [72] developed a series of self-assembling micelles based on star-shaped b-CD-caprolactone amphiphilic copolymers and subsequently, micellar carriers based on a folate conjugated CD-caprolactone polymer (FA-CD-PEL). In vitro and in vivo studies of FA-CD-PEL proved that they enhance the antitumor efficacy of doxorubicin while reducing its toxicity in non-cancer cells [73].
Table 2. A summary of in vitro and in vivo studies of drug carriers based on folateappended cyclodextrins. Cyclodextrin
EN-b-CD-FA [42]
Drug MTX
Cancer type In vitro - Breast (MCF-7) - Lung (A549)
EN-b-CD-FA [44]
DCX
In vitro -
Cervical (HeLa)
-
Oral (KB)
-
Liver (HepG2)
18
Observed effects - Lower (four times) cytotoxity towards normal cell lines and higher cytotoxity towards studied cancer lines (as compared to free MTX). Cytotoxity comparable to free DTX. Vialability of the cells decreases in the series OB > HepG2> HeLa > KB -
In vitro
b-CD-CA2-FA [51]
-
Oral (KB)
-
Lung (A549)
In vivo - Colon (mice inoculated with Colon26, cells, an FR-αpositive mouse colon carcinoma cell line)
a-CD-CA1-FA [52]
DOX
In vitro -
b-CD-CA1-FA [52]
DOX
In vitro -
g-CD-CA1-FA [52]
DOX
DOX
Oral (KB)
In vivo - Colon (mice inoculated with Colon26, cells, an FR-αpositive mouse colon carcinoma cell line) In vitro -
Me-b-CD-A [53]
Oral (KB)
Oral (KB)
In vitro -
Oral (KB)
19
cells expressing higher FR levels are more sensitive to formulations containing folate. Increase of DOX antitumor activity in KB Increase of DOX cellular uptake (2x) in KB Inhibition of DOX cellular efflux (KB) No change in antitumor activity in A549
Inhibition of tumor growth, as compared to control and DOX alone after intratumoral administration Inhibition of tumor growth after intravenous injection of the complex (contrary to lack of inhibition in case of free DOX injection) No change in anticancer activity as compared to free DOX
Increase of DOX cellular uptake Inhibition of DOX cellular efflux Increased antitumor activity, as compared to free DOX, after intratumoral and intravenous administration to tumor-bearing mice No change in anticancer activity as compared to free DOX Increased antitumor activity, as compared to free DOX Increase of DOX
cellular uptake No effect on DOX cellular efflux
In vivo Colon (mice inoculated with Colon-26, cells, an FR-α-positive mouse colon carcinoma cell line)
b-CD-s-Va-FA [54]
PCX
In vitro Breast cancer (T47D and ZR-75-1)
b-CD-p-Va-FA [54]
PCX
In vitro Breast cancer (T47D and ZR-75-1)
C60-g-CD-FA [56]
CBP
In vitro Cervical (HeLa)
b-CD-CPL-FA [58]
DOX7-ss-b-CD-FA7 [59]
DOX
DOX
In vitro -
Liver (HepG2)
-
Oral (KB)
-
Lung (A549)
In vitro -
Breast (mouse EMT6/P)
-
Breast (mouse, EMT6/AR1 - resistant to DOX)
-
Lung (A549)
20
Inhibition of tumor growth after intravenous injection of the complex Significant improvement of the mice survival rate, as compared to control (5% mannitol), free DOX and DOX/M-b-CD complex On T-47D cellscomparative anticancer efficacy to that of free PCX On ZR-75-1 cells significant improvement of anticancer efficacy on cells On T-47D cellscomparative anticancer efficacy to that of free PCX On ZR-75-1 cells significant improvement of anticancer efficacy on cells Increased antitumor activity, as compared to free CBP Increase of DOX cellular uptake in HepG2 and KB
Increased cellular uptake in EMT6/P and EMT6/AR1 cells Cellular uptake in A549 similar to that of free DOX Reduced efflux of DOX from EMT6/P and EMT6/AR1 cells No change in DOX efflux from A549 as
a-CD (G4)-FA [71]
siRNA
In vitro Oral (KB)
-
compared with that of free DOX - Antitumor activity was observed
In vivo -
Colon (mice inoculated with Colon26, cells, an FR-αpositive mouse colon carcinoma cell line)
-
-
FA-CD-PEL [73]
DOX
In vitro -
Cervical (HeLa)
-
Oral (KB)
-
Lung (A549)
In vivo - Oral (KB cellxenografted nude mouse model)
Inhibition of tumor growth No severe side effects under the experimental conditions Significant increase of the blood half-life of si RNA within the complex
Increased cellular uptake in HeLa cells and to the lesser extent in KB cells No change in cellular uptake in A549 cells - Significant improvement of the mice survival rate
4. Folate receptor targeting via other delivery agents. Comparison with folate appended cyclodextrins.
Apart from folate-based cyclodextrins there is a great number of other folate-based carriers described in the literature, including simple folate as well as liposome-folate conjugates and folate-decorated nanoparticles. All of the discussed carriers seem to be superior to the free drug due to the presence of the FA moiety which ensures FR targeting. On the other hand, folate moiety in the carrier competes with free the FA for the same receptor binding sites. The detailed analysis of advantages and disadvantages of specific drug delivery systems is presented in Table 2. Drug-folate conjugates are
21
designed by a covalently linking the drug molecule to a folate carrier through an easy to cleave spacer, which allows the drug to be released at the cellular target site. This approach, is usually time and money consuming. There is a large number of studies concerning delivery of chemotherapeutic agents with the application of liposomes. In 1998 Lee and Low reported the synthesis of folate-conjugated liposomes [74]. Later a number
of
data
concerning
such
derivatives,
including
folate-PEG-
distearoylphosphatidyletha-nolamine
[75],
folate-PEG-cholesterol,
folate-PEG-
cholesteryl [76], and folateglutathione-PEG-distearoyl-phosphatidylethanolamine [77], which improved FR targeted delivery of liposomes, were reported. Unfortunately, the folate moiety self-aggregates on the surface of the liposomes, which leads to limited FR targeting efficiency [78]. Many studies were dedicated to the application of folatedecorated nanoparticles [79, 80]. As with liposomes, PEG is often incorporated in their structure, in order to improve the solubility of the carrier and subsequently solubility and half-life of the drug. FA linked to nanoparticles ensures an extended circulation time and guides nanoparticle-based carriers to FR-positive tumors [81]. The huge drawback of such approach is an accumulation of FA-decorated nanoparticles in the liver and kidneys, which may result in damaging their proper functioning [82]. As FA-decorated nanoparticles compete with free FA for the same receptor binding sites, the development of nanoparticles which can bind more effectively to folate receptor is also a major issue. Addition of more FA groups on the nanopoparticle surface seems to be favorable; nevertheless there is an optimal amount of FA, above which the uptake of FAbased-nanocarriers decreases [83]. In general, inclusion complexes of drugs with CDs are characterized by better solubility in water and better drug bioavailability. Nevertheless, the use of only very stable host: guest complexes (Ka values of at least 104−105 M−1) may find a practical application, as weaker complexes rapidly dissociate into free CDs and free drugs after parenteral administration [84,85]. To conclude, all of the discussed carriers improve the activity of the drug, nevertheless application of each class of these molecules has some drawbacks, that still need to be addressed.
22
Table 3. Comparison of various folate-based carriers. Drug-delivery system
Advantages
Disadvantages
Drug-folate conjugate
Specific FR targeting Minimization of systemic distribution of the drug
The amount of a drug attached to the folate carrier (drug loading) is limited due to the available number of attachment sites. Development of the new conjugates and their subsequent synthesis is often time-consuming and expensive - Competition with free FA for the same receptor binding sites
Liposome-folate-encapsulated
Specific FR targeting Ability for encapsulation and delivery of a large quantity of a drug in a single carrier More efficient transport of the drug molecule leading to its better uptake and higher cytoxicity
Selfaggregation of folate on the surface of liposomes limits their potential application Liposome stabilizing factors, such as incorporating PEG chains into the structure, prevent efficient unloading of the drug which follows uptake by a target cell - Competition with free FA for the same receptor binding sites
Specific FR targeting Ability for encapsulation and delivery of a large quantity of a drug in a single carrier
- Significant amounts of FA-decorated nanoparticles accumulate in the liver and kidneys. Accumulation of FAdecorated nanoparticles containing chemotherapeutic drugs in the kidneys increases the risk of impairing their proper functioning - FA-decorated nanoparticles compete with free FA for the same receptor binding sites - Manufacturing issues (scalability and batch-to-batch consistency)
drug
Folate-decorated nanoparticles
Folate appended
-
Specific FR
23
-
The amount of
cyclodextrin/drug inclusion complex
targeting Increase of the water solubility of hydrophobic drugs Increase of the drug’s bioavailability
a drug inside a single carrier is limited due to the limited inclusion possibility (based on the complex stoichiometry) Competition with free FA for the same receptor binding sites - In vivo application: only complexes of Kas>104 M-1 do not dissolve
5. Conclusions. The application of monosubstituted cyclodextrins, CDs with a higher degree of substitution, as well as various polymers derivatized with folate, as targeted drug delivery systems was presented. The majority of conjugates are based on medium-sized b-CD, as it usually forms more stable complexes with studied guest compounds than other two native (a- and g-) CDs. Drugs such as methotrexate, b-estradiol, chlorambucil, scutellarin, 5-fluorouracil, doxorubicin, paclitaxel, and carboplatin, in most cases formed 1:1 inclusion complexes. The comparison of the stability of the drug/FA-appended cyclodextrin complexes with the corresponding complexes based on a parent unsubstituted cyclodextrin proved that, in most cases, FA moiety covalently linked to the cyclodextrin core improves the stability of the complex. It is most pronounced for doxorubicin complexes (Ka three orders of magnitude higher for Me-b-CD-FA and four orders of magnitude higher for b-CD-CA1-FA and b-CD-CA2-FA than for native b-CD). Synthetic procedures leading to FA-CD conjugates were presented in detail. The majority of the complexes were studied only in vitro, with only a number studied also in vivo. In vitro studies were usually performed in FR-positive cells and often compared with FR-negative cells. The results obtained from these studies suggest the superior properties of the studied systems as compared to the free drug, mainly in the improved anticancer activity, increased drug cellular uptake, and limited drug cellular efflux. Folate appended cyclodextrins were also found to be good sustained release systems for DNA and siRNA. Comparison of the folate-appended cyclodextrins with other FA-based drug24
carriers including simple folate, liposome-folate conjugates, and folate-decorated nanoparticles was also performed. All of the discussed carriers improve the activity of the drug; nevertheless an application of each class of these molecules has several drawbacks, that still need to be addressed. Definitely, the application of cyclodextrins avoids the risk of disadvantages characteristic to other carriers, such as accumulation of FA-decorated nanoparticles containing chemotherapeutic drugs in kidneys leading to impairment of their proper functioning, or manufacturing issues (scalability and batch-tobatch consistency). Nevertheless, the necessity of the application of only very strong complexes (of Ka values of at least 104−105 M−1) to maintain their non-dissociated form in vivo after parenteral administration, creates a great challenge for the design and efficient synthesis of proper CD-based carriers. At the moment the clinical applications of folateappended cyclodextrins for drug, DNA and siRNA are still at prospects of the future. Further investigations are required regarding pharmacokinetics, and safety profiles, along
with
detailed
understanding
of
mechanisms
by which
folate-appended
cyclodextrins enhance antitumor activity of chemoterapeutic drugs. Nevertheless, these new carriers make a very promising class of FR-a targeting delivery systems.
Acknowledgements This work was financed by the Polish Ministry of Science and Higher Education, Grant No. IP2012007472. References: [1] C.S. Kue, A. Kamkaew, K. Burgess, L.V. Kiew, L.Y. Chung, H.B. Lee, Med. Res. Rev. 2016, 494. [2] Y. Zhong, F. Meng, C. Deng, Z. Zhong, Biomacromol, 2014, 15, 1955. [3] A.M. Gazzali, M. Lobry, L. Colombeau, S. Acherar, H. Azaïs, S. Mordon, P. Arnoux, F. Baros, R. Vanderesse, C. Frochot, Eur. J. Pharm. Sci. 2016, 93, 419. [4] C. Chen, J. Ke, X.E. Zhou, W. Yi, J.S. Brunzelle, J. Li, E-L. Yong, H.E. Xu, K. Melcher, Nature, 2013, 500,486. [5] X. Guo, C.L. Shi, J. Wang, S.B. Di, S.B. Zhou, Biomater., 2013, 34, 4554. 25
[6] B. Sahoo, K.S.P. Devi, R. Banerjee, T.K. Maiti, P. Pramanik, D. Dhara, ACS Appl. Mater. Interfaces, 2013, 5, 3884. [7] Chaudhury, S. Das, Curr. Pharm. Biotechnol. 2015, 16, 333. [8] S. Santra, C. Kaittanis, O.J. Santiesteban, J.M. Perez, J. Am. Chem. Soc., 2011, 133, 16680. [9] J.W. Lee, J.Y. Lu, P.S. Low, P.L. Fuchs, Bioorg. Med. Chem., 2002, 10, 2397. [10] W.A. Henne, D.D. Doorneweerd, A.R. Hilgenbrink, S.A. Kularatne, P.S. Low, Bioorg. Chem. Lett., 2006, 16, 5350. [11] I.R. Vlahov, C.P. Leamon, Bioconjugate Chem., 2012, 23,1357. [12] A. Guaragna, A. Chiaviello, C. Paolella, D. D’Alonzo, G. Palumbo, G. Palumbo, Bioconjugate Chem., 2012, 23, 84. [13] Y. Singh, M. Palombo, P.J. Sinko, Curr. Med. Chem., 2008, 15, 1802. [14] J. Szejtli, Chem. Rev., 1998, 98, 1743. [15] M.E. Brewster, T. Loftsson, Adv. Drug Deliv. Rev., 2010, 59, 645. [16] M. Ceborska, Curr. Org. Chem., 2014, 18, 1878. [17] H-J. Schmannan, E. Schollmeyer, J. Cosmet. Sci., 2002, 53, 185. [18] G. Astray, C. Gonzalez-Barreiro, J.C. Mejuto, R. Rial-Otero, J. Simal-Gandara, Food Hydrocolloid., 2009, 23, 1631. [19] J. Yin, Z.W. Zhou, S.F. Zhou, Drug Deliv. Transl. Res., 2013, 3, 364. [20] T. Irie, E. Uekama, J. Pharm. Sci., 1997, 86, 147. [21] T. Loftsson, M.E. Brewster, J. Pharm. Pharmacol., 2010, 62, 1607. [22] J. Zhang, P.X. Ma, Adv. Drug Deliv. Rev. 2013, 65, 1215. [23] F.M. Sirotnak, B.Tolner, Annu. Rev. Nutr., 1999, 19, 91. [24] D.W. Horne, K.A. Reed, J. Hoefs, H.M. Said, Am. J. Clin. Nutr., 1993, 58, 80. [25] Y.G. Assaraf, C.P. Leamon, J.A. Reddy, Drug Resist. Updat., 2014, 17, 89. [26] J. Shen, K.S. Putt, D.W. Visscher, L. Murphy, C. Cohen, S. Singhal, G. Sandusky, Y. Feng, D.S. Dimitrov, P.S., Oncotarget, 2015, 6, 14700. [27] S.D. Weitman, A.G. Weinberg, L.R. Coney, V.R. Zurawski, D.S. Jennings, B.A. Kamen, Cancer Res., 1992, 52, 6708.
26
[28] D.J. O’Shannessy, E.B. Somers, E. Albone, X. Cheng, Y.C. Park, B.E. Tomkowicz, Y. Hamuro, T.O. Kohl, T.M. Forsyth, R. Smale, Y-S. Fu, N.C.Nicolaides, Oncotarget. 2011, 2, 1227. [29] H. Kurahara, S. Takao, T. Kuwahata, T. Nagai, Q. Ding, K. Maeda, H. Shinchi, Y. Mataki, K. Maemura, T. Matsuyama, S. Natsugoe, Ann. Surg. Oncol. 2012, 19, 2264. [30] J. Shen, A.R. Hilgenbrink, W. Xia, Y. Feng, D.S. Dimitrov, M.B. Lockwood, R.J. Amato, P.S. Low, J. Leukoc. Biol., 2014, 96, 563. [31] Y. Tian, G. Wu, J.C. Xing, J. Tang, Y. Zhang, Z.M. Huang, Z.C. Jia, R. Zhao, Z.Q. Tian, S.F. Wang, X.L. Chen, L. Wang, Y.Z. Wu, B. Ni, BMC Immunol., 2012, 13, 30. [32] E.I. Sega, P.S. Low, Cancer Metastasis Rev., 2008, 27, 655. [33] L. Teng, J. Xie, L. Teng, R.J. Lee, Expert. Opin. Drug. Deliv., 2012, 9, 901. [34] H. Shi, J. Guo, C. Li, Z. Wang, Drug Des. Devel. Ther., 2015, 9, 4989. [35] K. Chmurski, P. Stępniak, J. Jurczak, Carbohydr. Polym., 2016, 138, 8. [36] M. Ceborska, M. Zimnicka, M. Pietrzak, A. Troć, M. Koźbiał, J. Lipkowski, Org. Biomol. Chem., 2012, 10, 5186. [37] M. Zimnicka, A. Troć, M. Ceborska, M. Jakubczak, M. Koliński, W. Danikiewicz, Anal. Chem., 2014, 86, 4249. [38] M. Ceborska, M. Zimnicka, M. Wszelaka-Rylik, A Troć, J. Mol. Struc., 2016, 1109, 114. [39] A. Clementi, M.C. Aversa, C. Corsaro, J. Spooren, R. Stancanelli, C O’Connor, M. McNamara, A. Mazzaglia, J. Incl. Phenom. Macrocycl. Chem., 2011, 69, 321. [40] Z. Tofzikovskaya, C. O’Connor, M. McNamara, J. Incl. Phenom. Macrocycl. Chem., 2012, 74, 437. [41] Z. Tofzikovskaya, A. Casey, O. Howe, C. O’Connor, M. McNamara, J. Incl. Phenom. Macrocycl. Chem., 2015, 81, 85. [42] B. Yang, L. Zhao, X. Yang, X.L. Liao, J. Jang, J.H. Zhang, C.Z. Gao, Carbohydr. Polym., 2013, 92, 1308. [43] R. Liao, Y. Zhao, X. Liao, M. Liu, C. Gao, J. Yang, B. Yang, Polym. Adv. Technol., 2015, 26, 487. [44] J. Xu, B. Xu, D. Shou, F. Qin, Y. Xu, Y. Hu, Eur. J. Pharm. Sci., 2016, 83, 132. 27
[45] H. Yao, S.S. Ng, W. O. Tucker, Y-K-T. Tsang, K. Man, X-M. Wang, B.K.C. Chow, HF. Kung, G-P. Tang, M.C. Lin, Biomater., 2009, 30, 5793. [46] Y. Zhou, H. Wang, C. Wang, Y. Li, W. Lu, S. Chen, J. Luo, Mol. Pharm., 2012, 9, 1067. [47] P. Calicetti, S. Salmaso, A. Semenzato, T. Carofiglio, R. Fornasier, M. Fermeglia, M. Ferrone, S. Pricl, Bioconjugate Chem., 2003, 14, 899. [48] S. Salmaso, A. Samenzato, P. Caliceti, J. Hoebeke, F. Sonvico, C. Dubernet, P. Couvreur, Bioconjugate Chem., 2004, 15, 997. [49] H. Zhang, Z. Cai, Y. Sun, F. Yu, Y. Chen, B. Sun, J. Biomed. Res. Mater. Part A, 2012, 100A, 2441. [50] S. Salamaso, S. Bersani, A. Semenzato, P. Caliceti, J. Drug Target, 2007, 15, 379. [51] A. Okamatsu, K. Motoyama, R. Onodera, T. Higashi, T. Koshigoe, Y. Shimada, K. Hattori, T. Takeuchi, H. Arima, Bioconjugate Chem., 2013, 24, 724. [52] A. Okamatsu, K. Motoyama, R. Onodera, T. Higashi, T. Koshigoe, Y. Shimada, K. Hattori, T. Takeuchi, H. Arima, Biomacromol., 2013, 14, 4420. [53] K. Motoyama, R. Onodera, A. Okamatsu, T. Higashi, R. Kariya, S. Okada, H. Arima, J. Drug. Target., 2014, 22, 211. [54] N. Erdogar, G. Esendagli, T.T. Nielsen, M. Sen, L. Oner, E. Bilensoy, Int. J. Pharm. 2016, 509, 375. [55] M. Agueros, V. Zabaleta, S. Espuelas, M.A. Campanero, J.M. Irache, J. Control. Release, 2010, 145, 2. [56] Y. Liu, Y. Pu, L. Sun, H. Yao, B. Zhao, R. Zhang, Y. Zhang, J. Biomed. Nanotechnol., 2016, 12, 1393. [57] C. Su, H. Li, Y. Shi, G. Wang, L. Liu, L. Zhao, R. Su, Int. J. Pharm., 2014, 474, 202. [58] Q. Zhou, X. Guo, T. Chen, Z. Zhang, S. Shao, C. Luo, J. Li, S. Zhou, J. Phys. Chem. B, 2011, 115, 12662. [59] H. Mizusako, T. Tagami, K. Hattori, T. Ozeki, J. Pharm. Sci., 2015, 104, 2934. [60] J. Yang, H. Chen, I.R. Vlahov, J.X. Cheng, P.S. Low, Proc. Natl. Acad. Sci. U.S.A., 2006, 103, 13872. [61] F. Pattarino, L. Giovannelli, G.B. Giovenzana, M. Rinaldi, M. Trotta, J. Drug Deliv. Sci. Technol., 2005, 15, 465. 28
[62] B. Yang, L.-J. Yang, J. Lin,Y. Chen,Y. Liu, J. Incl. Phenom. Macrocycl. Chem., 2009, 64, 149. [63] R. Anand, S. Ottani, F. Manoli, I. Manet, S. Monti, RSC Adv.,2012, 2, 2346. [64] M.A. Mintzer, M.W. Grinstaff, Chem. Soc. Rev., 2011, 40, 173. [65] H. Arima, K. Motoyama, T. Higashi, Adv. Drug Deliv. Rev., 2013, 65, 1204. [66] H. Arima, K. Motoyama, T. Higashi, Curr. Top. Med. Chem., 2014, 14, 465. [67] T. Tsutsumi, F. Hirayama, K. Uekama, H. Arima, J. Controlled Release, 2007, 119, 349. [68] H. Arima, T. Tsutsumi, A. Yosshimatsu, H. Ikeda, K. Motoyama, T. Higashi, F. Hirayama, K. Uekama, Eur. J. Pharm. Sci., 2011, 44, 375. [69] H. Arima, A. Yoshimatsu, H. Ikeda, A. Ohyama, K. Motoyama, T. Higashi, A. Tsuchiya, T. Niidome, Y. Katayama, K. Hattori, T. Takeuchi, Mol. Pharm., 2012, 9, 2591. [70] F. Kukowska-Latallo, A.U. Bielinska, J. Johnson, R. Spindler, D.A. Tomalia, J.R. Baker, Jr., Proc. Natl. Acad. Sci. U.S.A., 1996, 93, 4897. [71] A. Ohyama, T. Higashi, K. Motoyama, H. Arima, Bioconjugate Chem., 2016, 27, 521. [72] L.Y. Qiu, R.J. Wang, C. Zheng, Y. Jin, Nanomedicine, 2010, 5, 193. [73] L. Zhang, J. Lu, Y. Jin, L. Qiu, Colloids Surf. B Biointerfaces, 2014, 122, 260. [74] R.J. Lee, P.S. Low, BBA. Biomembranes, 1995, 1233, 134. [75] A. Gabizon, A.T. Horowitz, D. Goren, D. Tzemach, F. Mandelbaum-Shavit, M.M. Qazen, S. Zalipsky, Bioconjugate Chem. 1999, 10, 289. [76] W. Guo, T. Lee, J. Sudimack, R.J. Lee, Liposome Res., 2000, 10,179. [77] G. Xiang, J. Wu, Y. Lu, Z. Liu, R.J. Lee, Int. J. Pharmaceut., 2008, 356, 29. [78] Y. Liu, S. Xu2, L. Teng, B. Yung, J. Zhu, H. Ding, R.J. Lee, Anticancer Res., 2011, 31, 1521. [79] L. Brannon-Peppas, J.O. Blanchette, Adv. Drug Deliv. Rev., 2012, 64, 206. [80] L. Xu, Q. Bai, X. Zhang , H. Yang, J. Control. Release, 2017, 252, 73. [81] C. Yue, P. Liu, M. Zheng, P. Zhao, Y. Wang, Y. Ma, L. Cai, Biomater., 2013, 34, 6853. [82] J.S. Kim, M.H. Oh, J.Y. Park, T.G. Park, Y.S. Nam, Biomater., 2013, 34, 2370. 29
[83] N.V. Nukolova, H.S. Oberoi, S.M. Cohen, A.V. Kabanov, T.K. Bronich, Biomater., 2011, 32, 5417. [84] V.J. Stella, V.M. Rao, E.A. Zannou, V.V. Zia, Adv. Drug Deliv. Rev., 1999, 36, 3. [85] V.J. Stella, Q. He, Toxicol. Pathol., 2008, 36, 30.
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