Letter pubs.acs.org/macroletters
Controlled Synthesis of End-Functionalized Mannose-6-phosphate Glycopolypeptides for Lysosome Targeting Soumen Das,* Nimisha Parekh, Basudeb Mondal, and Sayam Sen Gupta* CReST Chemical Engineering Division, CSIR National Chemical Laboratory, Dr. Homi Bhabha Road, Pune 411008, India S Supporting Information *
ABSTRACT: The ubiquitous expression of the mannose-6phosphate receptor on the majority of human cells makes it a valid target in the quest to deliver therapeutics selectively to the lysosome. In this work end-functionalized polyvalent mannose-6-phosphate glycopolypeptides (M6P-GPs) with high molecular weights (up to 22 kDa) have been synthesized via NCA polymerization. These synthetic M6P-GPs were found to display minimal toxicity to cells in vitro and show exceptional selectivity for trafficking into lysosomes in various cell lines. Comparison of the cellular uptake behavior of M6PGP and the corresponding mannose-GP polymer reveals that incorporation of the phosphate moiety at the 6-position of mannose completely alters its trafficking behavior and becomes exclusively lysosome specific. We also demonstrate that trafficking of M6P-GPs in mammalian cells is likely associated with the CI-MPR receptor pathway.
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doses of these enzymes need to be administered to achieve significant therapeutic efficacy.11 Modification of lysosomal enzymes with higher amounts of M6P residues for enhanced targeted delivery remains a critical challenge.12 Previous studies demonstrate that CI-MPR exists as a dimer in the membrane which allows for high affinity binding of ligands that are multivalent.13−17 The enhanced binding affinity of CI-MPR to glycoproteins that contain multiple M6P residues (Kd = 2−20 nM) in comparison to monomeric M6P (Kd = 7 μM) is attributed to the so-called “glycoside cluster effect”.18−20 Additionally, the structure of free CI-MPR and its ligand-bound state suggest that the presence of flexibly spaced M6P units would lead to higher binding affinity.20 The many-fold enhancement for the affinity and selectivity of M6P-containing ligands toward the CI-MPR receptor has prompted the scientific community to investigate synthetic ligands that can present multiple copies of M6P moieties simultaneously to facilitate more efficacious targeting.21−25 Recently, Overkleeft et al. have demonstrated the synthesis of a multivalent (oligomeric) M6P ligand for the CI-MPR targeting and trafficking through the endolysosomal pathway.21 However, tedious synthetic steps associated with the solid-phase peptide synthesis approach, lack of polyvalency (polymeric), and incorporation of several non-natural triazole moieties on the ligand backbone would limit their function as a natural mimic. Therefore, we envisaged that it would be interesting to
n mammalian cells, mannose-6-phosphate receptors play a critical role in the sorting of lysosomal enzymes from secretory proteins and their subsequent delivery into lysosomes.1 These receptors, which include the ∼300 kDa insulin-like growth factor-II (IGF-II)/cation-independent (CI) multifunctional transmembrane glycoprotein mannose-6-phosphate (M6P) receptor (CI-MPR), bind lysosomal enzymes containing phosphomannosyl residues in the trans-golgi network (TGN) and transport them to endosomes where the low pH leads to the dissociation of the ligand−receptor complex.2 The receptors then return to the TGN to repeat another round of this process. As a result, the receptor CI-MPR is largely localized in the intracellular compartments and shuttles between TGN and endosomes with ∼10% of the receptor being present on the cell surface.3 Additionally, the CIMPR receptor is overexpressed in the early stage of several cancers, particularly in breast and prostate cancer, and hence can serve as an early marker for these cancers.4,5 The ability of CI-MPR to deliver cargo specifically to the lysosome can thus be selectively targeted using M6P-labeled carriers to dispatch cytotoxic drugs inside lysosomes for destroying cancer cells.6−8 In addition, CI-MPR can be targeted to deliver M6P-labeled enzymes into lysosomes for enzyme replacement therapies (ERTs) in non-neural lysosomal storage disorders (LSDs).9,10 LSDs are a group of more than 40 metabolic disorders that result from the deficient activity of specific lysosomal enzymes inside the lysosomes leading to progressive accumulation of its substrate inside the cell. Nowadays several M6P-modified enzymes are used clinically for the treatment of LSD. However, the low levels of M6P modification dramatically reduce its binding efficiency to the receptor, and hence relatively high © XXXX American Chemical Society
Received: April 18, 2016 Accepted: June 13, 2016
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DOI: 10.1021/acsmacrolett.6b00297 ACS Macro Lett. 2016, 5, 809−813
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ACS Macro Letters
simultaneous oxidation to form the corresponding phosphate (Scheme 1). Thus, the synthesis of carboxylic acid 4 from allyl 2,3,4,6-tetra-O-acetyl-α-D-mannopyranoside involved a facile multistep reaction with an acceptable overall yield of 64%. The carboxylic acid 4 was used in the DIPC coupling reaction with 9-BBN-protected L-lysine to afford the desired conjugate 5 (Scheme 2). Deprotection of the 9-BBN moiety of
synthesize high molecular weight M6P-glycopolypeptides (Figure 1) having pendant M6P moieties in the side chain as
Scheme 2. Synthesis of M6P-Glycopolypeptidesa
Figure 1. Chemical structures of M6P-glycopolypeptides.
a natural mimic of proteins bearing M6P moieties. Such a glycopolypeptide can help us understand the complexity and individuality of CI-MPR trafficking for lysosomal targeting. In addition, as demonstrated by us and others, amphiphilic glycopolypeptides can be self-assembled into vesicles, micelles, and nanorods.26−29 These self-assembled nanostructures synthesized from M6P-glycopolypeptide can be used to encapsulate therapeutics or lysosomal enzymes for targeting and delivering them exclusively to lysosomes. Herein, we describe the synthesis of an end-functionalized M6P-glycopolypeptide (M6P-GP) with the aim of delivering therapeutics inside the lysosomes selectively. The design involves placement of a negatively charged M6P group on a poly-L-lysine backbone such that it would maintain the required flexibility for multivalent binding to its corresponding receptor with high affinity. Further, end-functionalized M6PGPs can allow easy attachment strategies to various proteins and enzymes for therapeutic applications. While designing the M6P-GP synthesis, routes involving polymerization of M6P containing α-NCA and installation of M6P moiety postpolymerization were considered.30 Although glycopolypeptides have been synthesized by post polymerization methods,31−34 they do not ensure 100% glycosylation density on the side chains. To enable a formation of 100% glycosylated M6P-GPs, we undertook an approach that combined the preparation and polymerization of a M6P-functionalized NCA monomer.35−38 Such a synthetic strategy also allows synthesis of both random and block copolymers with M6P-GP. The principle challenge of this methodology is associated with the orthogonal protection/ deprotection strategies of the phosphate and carbohydrate moieties during the synthetic course. In our synthetic strategy, we used the benzyl protection of phosphates and acetyl protection of carbohydrates as is shown in Scheme 1. The 6-
a
(a) DIPC, DMAP, THF, rt, 6 h (75% yield); (b) CHCl3:CH3OH, rt, 24 h; (c) Triphosgene, N-methylmorpholine, THF, 55 °C, 1 h (58% yield); (d) n-hexylamine, proton sponge, DMF (∼95% yield); (e) TFA-DCM or H2, Pd−C (98% yield); (f) Hydrazine hydrate, MeOH, rt (85% yield).
the conjugate 5 was achieved by stirring this compound in a mixture of chloroform and methanol at room temperature for 24 h. Resulting amino acid 6 was treated with triphosgene and N-methylmorpholine in THF at 55 °C to obtain 6-deoxy-6dibenzylphosphate-glyco-NCA monomer (Scheme 2) which was repurified by precipitation and anhydrous flash column chromatography (yield 58%).39 The FT-IR spectrum of the resulting NCA displayed two characteristic anhydride stretchings at 1784 and 1854 cm−1 (SI Figure 1). Ring-opening polymerization (ROP) of α-NCA with a primary amine initiator proceeded to completion at room temperature to afford the corresponding M6P-GPs in excellent yield with no detectable side products (Scheme 2). The resulting glycopolypeptides were purified by reprecipitation, and their number-average molecular weights (Mn) were estimated by 1 H NMR (SI figure: NMR section). Mn was calculated from the relative intensity of the peak at 0.85 ppm due to the characteristic proton present in the initiator hexylamine (alkane −CH3) with the proton peaks of the benzyl group (−C6H5) present in the dibenzylphosphate moiety of the polymer (7.33 ppm). The molecular weight distributions observed from GPC were monomodal (SI Figure 2), and the PDI obtained was reasonably narrow and ranged from 1.05 to 1.18 (Table 1). In addition to that, the obtained molecular weight corroborated well with the targeted monomer/initiator ratio (Table 1) suggesting control over polymer chain length during polymerization. To obtain “clickable” end-functionalized M6P-GP, the polymerization was carried out with azido-PEG-NH2 as the initiator. The resultant polymer displayed a sharp peak at 2108 cm−1 in FT-IR that is characteristic of the organo azide stretch indicating that the initiator was incorporated into the main polypeptide chain (SI Figure 3). The synthesis of such “clickable” M6P-GPs is extremely useful for their subsequent conjugation with biological entities for cellular uptake experiments.
Scheme 1. Synthesis of 6-Deoxy-6-dibenzylphosphate-2,3,4tri-O-acetyl α-D-Mannopyranoside Ethanoic Acida
a
(a) ACN−CCl4−H2O; RuCl3·xH2O, NaIO4, rt, 2 h (82% yield).
OH group of the allyl 6-deoxy-6-hydroxy-2,3,4-tri-O-acetyl α-Dmannopyranoside 1e (SI Scheme 1) was phosphorylated using phosphoramidite reagent 2b (SI Scheme 2) which allows the incorporation of precursor phosphite at the 6-position in near quantitative yield (Scheme 1). Allyl 6-deoxy-6-dibenzylphosphite-2,3,4-tri-O-acetyl α-D-mannopyranoside 3 was oxidized to the corresponding carboxylic acid 4 using NaIO4 and RuCl3 in acetonitrile during which the phosphite group also underwent 810
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ACS Macro Letters Table 1. Synthesis of M6P-GPs at Different Monomer to Initiator Ratio M/Ia
Mnb (× 10−3 g/mol)
Mw/Mnc
DPd
yield (%)e
15 30 40
11.6 23.2 30.1
1.05 1.16 1.18
16 32 43
92 94 94
a
M/I = monomer to initiator ratio [initiator (I) is hexylamine]. Calculated from gel permeation chromatography in DMF containing 0.025 M LiBr as eluent at 60 °C. dDegrees of polymerization (DP) were calculated from 1H NMR. eTotal isolated yield of the M6P-GPs. b,c
The labile benzyl-protecting groups on the phosphate moiety were removed by using both TFA-DCM or by hydrogenation depending on the presence of the end group in the M6P-GPs. The deprotection of the acetate group of carbohydrates was performed using hydrazine hydrate in MeOH at RT, and the resultant polymer was purified by dialysis. 31P NMR of watersoluble M6P-GPs displays a broad peak at −0.38 ppm suggesting that the phosphate groups remain intact in the glycopolypeptide (SI figure: NMR section). The secondary conformation of M6P-GPs was examined in aqueous media using circular dichroism (CD) spectroscopy. The CD spectra were consistent with an α-helical chain conformation over a broad pH range of 4.0−11.0. (SI Figure 4). Therefore, introduction of the charged phosphate group 14 atoms from the peptide backbone had no effect on the secondary conformation as has been shown earlier.40 Preliminary cellular entry assays were conducted to ascertain whether the M6P-GP was able to selectively traffic into lysosomes. Since cell viability of the polymers is a precondition for such trafficking studies, we first performed a cytotoxicity test (MTT assay) of fully deprotected 30-M6P-GP (9b) with MDA-MB-231, L929, and MCF-7 cell lines (SI Figure 5). Results of the MTT assay demonstrate that M6P-GPs exhibit minimal toxicity to the cell in vitro since 80% of the cell remains viable up to 500 μg of 9b after 48 h incubation. To visualize the entry of M6P-GP upon cellular internalization, water-soluble fluorescein labeled 30-M6P-GP (FL-9b) was synthesized by reaction of protected 30-M6P-GP (7b) with fluorescein-NHS (FL-NHS) followed by deprotection of carbohydrate and phosphate (SI Scheme 3). Lysosomes are the major intracellular acidic compartments, and their numbers and acidity could significantly increase in cancer cells. To visualize the potential utility of M6P-GPs for lysosomal targeting, FL-9b (200 μg/mL) was incubated with MDA-MB-231 breast cancer cells for 4 h at 37 °C and then treated with LysoTracker Red DND-99 to specifically stain acidic lysosomes. Fluorescence microscopy images show a large number of highly green fluorescent punctate vesicle-like structures inside the cells which are probably indicative of cellular organelles such as lysosomes (Figure 2). Further insights into the location of FL-9b inside the cells were obtained from the fluorescence distribution of FL-9b and LysoTracker Red. The distribution of fluorescence inside the cells exhibits colocalization of green fluorescence from FL-9b with LysoTracker Red dye (Figure 2), which indicates that the FL-9b was located inside the acidic lysosomes. In addition to that, the color intensity profile from the bright area of the merged images displays identical variation of color intensity with distance for both the green and red detection channels, which further supports the hypothesis that both the dyes are
Figure 2. Lysosome targeting with M6P-GP (FL-9b): MDA-MB-231 (a−d), L929 (e−h), and MCF-7 (i−l) cells were cultured for 4, 6, and 2 h, respectively, with FL-9b (200 μg/mL) in DMEM and then stained with LysoTracker Red (50 nM) for 30 min. The cells were probed by fluorescence microscopy. Merging of the FL signal (shown in green) and that of LysoTracker Red (shown in red) revealed colocalization as indicated by the yellow spots/areas (bars, 10 μm); cyan color arrow indicates punctate-like vescicles.
colocalized within the individual cellular organelles (SI Figure 6b). These experiments demonstrate that upon uptake of FL9b by MDA-MB-231 they are specifically trafficked to the lysosomal compartments. The targeting of FL-9b to lysosomes was then studied in noncancerous fibroblast L929 cells. The purpose for using a noncancerous L929 cell line was to examine how effectively M6P-GPs can be trafficked selectively toward lysosomes of a normal cell line. This is important since cells affected with LSD do not have CI-MPR receptors overexpressed on its surface unlike most cancer cells. It is therefore important to probe the efficiency of uptake of the M6P-GPs in normal cell lines to evaluate its therapeutic potential. As was observed in MDAMB-231 cells, localized intracellular green fluorescence bright spots were also colocalized with LysoTracker Red (Figure 2; SI Figure 7). Finally, FL-9b displayed rapid cellular uptake on the MCF-7 breast cancer cell line, known to overexpress a significant amount of CI-MPR receptors.6 Rapid cellular endocytosis was observed within 2 h at 200 μg/mL concentration (Figure 2; SI Figure 8) with subsequent trafficking to the lysosomes similar to what was observed for MDA-MB-231 and L929. The quantification of fluorescence intensity present inside the cells reveals ∼3-fold higher uptake in MCF-7 cells compared to MDA-MB-231 and L929 (SI Figure 9) which correlates well with the increased M6P receptors present on MCF-7 cells. This again suggests that M6P-GP is likely uptaken by the overexpressed CI-MPR receptors present on the MCF-7 cell surface and subsequently transferred into the lysosomes. To investigate the engagement of the CI-MPR receptor during cellular uptake of M6P-GP, competition assays were carried out on the MCF-7 cell line using free monomeric mannose-6-phosphate (M6P; SI Figures 10 and 11). Treatment of MCF-7 cells with 1.0 mM monomeric M6P for 1 h at 37 °C prior to addition of FL-9b (200 μg/mL) and subsequent incubation for another 2 h at 37 °C displays 80% reduction in the uptake of FL-9b (Figure 3). This finding highlights that the 811
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ACS Macro Letters
new M6P-GP could open up numerous potential therapeutic applications via targeting cell surface CI-MPR receptors.
CI-MPR receptors represent the predominant pathway available for uptake of M6P-GP.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmacrolett.6b00297. Synthetic procedures for all monomers and polymers with characterization by 1H, 13C, and 31P NMR, FT-IR, fluorescence spectra, CD spectra, and cellular experiment data (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. *E-mail:
[email protected].
Figure 3. Competition assay for the uptake of M6P-GP onto MCF-7 cells: (a) fluorescence microscopy image of MCF-7 cells treated with FL-9b, (b) fluorescence microscopy image of MCF-7 cells that were first pretreated with 1.0 mM monomeric M6P followed by FL-9b addition, and (c) normalized fluorescence intensity analysis after cellular uptake of FL-9b with and without monomeric M6P treatment. Green color shows internalized FL-9b; nucleus was stained with DAPI.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors acknowledge Prof. M G Finn for critically reading the manuscript and providing valuable suggestions and Mr. Srikanth Khake and Dr. P Rajmhanan for NMR support. We acknowledge Dr. Sudipta Basu for providing LysoTracker Red. SD and BM thank CSIR for a research fellowship. SSG acknowledges CSIR Network Project CSC0302 and CSC0134 for financial support.
Recently we reported the uptake of mannosylated glycopolypeptide nanoassemblies toward MDA-MB-231 cells in which ∼45% of the cellular uptake occurred through the overexpressed mannose-specific MRC2 receptor.41 This finding prompted us to study the characteristic differences between the trafficking behavior of both M6P-GP and mannose-GP in the MCF-7 cell line (since it overexpressed both M6P-specific CIMPR and mannose-specific MRC2 receptors).42,43 The cells treated with FL-mannose-GP displayed more evenly distributed fluorescence intensity inside the cells (SI Figure 12a) and were colocalized sporadically with LysoTracker Red (SI Figure 13). This is in contrast to FL-9b where the bright fluorescent spots inside the cell colocalized (SI Figure 12b) with LysoTracker Red almost precisely (SI Figure 13). This result establishes that trafficking of M6P-GP principally follows the CI-MPRmediated endolysosomal pathway. Together these observations imply that the incorporation of the phosphate moiety at the 6position of mannose completely altered its trafficking behavior and becomes mostly lysosome specific. This remarkable selectivity of M6P-GP toward lysosomes suggests that it can have more efficacious delivery applications toward lysosomes. In summary, end-functionalized M6P-GPs with pendant M6P moieties have been developed for efficient lysosomal targeting. The synthetic methodology developed by us allows facile synthesis of water-soluble M6P-GPs with high molecular weights. These synthetic M6P-GPs were also found to display minimal toxicity to cells in vitro. Cellular uptake experiments reveal that M6P-GPs selectively trafficked into lysosomes using the cell surface CI-MPR receptor. To our knowledge it represents the first example of a synthetic polyvalent ligand that mimics proteins conjugated with phosphomannosyl residues which is selectively trafficked into the lysosomes. The conjugation of this end-functionalized M6P-GP to other biological entities, such as recombinant lysosomal enzymes, may thus be a suitable alternative for delivery of these enzymes into cells affected by LSD. Moreover, such a synthetic methodology can be extended to synthesize amphiphilic M6P-GPs that can be self-assembled into soft nanomaterials for possible use as carriers in drug delivery. We believe that this
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