Dendritic macromers as in situ polymerizing biomaterials for securing cataract incisions

Article abstract of DOI:10.1021/ja045870l

Dendritic macromers are attractive macromolecules for hydrogel formation since high cross-linking densities at low polymer concentration can be obtained, varied physical properties can be observed based on the macromer structure, and low viscous aqueous s

Full text of DOI:10.1021/ja045870l

Published on Web 09/21/2004
Dendritic Macromers as in Situ Polymerizing Biomaterials for Securing
Cataract Incisions
Michel Wathier,† Pil J. Jung,‡ Michael A. Carnahan,‡ Terry Kim,‡ and Mark W. Grinstaff*,†
Departments of Biomedical Engineering and Chemistry, Metcalf Center for Science and Engineering,
Boston UniVersity, Boston, Massachusetts 02215, and Department of Ophthalmology,
Duke UniVersity Medical Center, Durham, North Carolina 27710
Received July 9, 2004; E-mail: mgrin@bu.edu
Dendritic macromolecules are versatile materials for medical
applications since the chemical composition, structure, and molec-
ular weight can be precisely controlled.1-7 Moreover, the hyper-
branched globular structure permits diverse and extensive surface
functionalization, which facilitates subsequent intermolecular cross-
linking, surface adhesion, or biological recognition. Cross-linkable
dendritic macromolecules are known to form macroscopic gels,
including hydrogels.8 Hydrogels are of general interest and are used
in applications ranging from drug delivery to wound healing.9,10
We are exploring the latter application using dendritic polymers
that are derived from monomers known to be natural metabolites
or biocompatible.8,11-13 The advantages of dendritic macromers for
hydrogel formation include high cross-linking densities at low
polymer concentration, varied physical properties through judicious
choice of the macromer structure, and low viscous aqueous solutions
for injection in an in vivo site of irregular shape for subsequent
cross-linking to form a well-integrated polymer network. Recently,
we have successfully repaired full-thickness 4.1-mm corneal
lacerations in enucleated eyes and chicken eyes in vivo using a
hydrogel sealant produced from photo-cross-linkable poly(glycerol-
succinic acid)-poly(ethylene glycol) hybrid dendritic-linear
macromolecules.13,14 We are currently investigating alternative
cross-linking chemistries to light initiation that spontaneously afford
a hydrogel within a few minutes at room temperature and at neutral
pH. Herein, we report the synthesis of peptide dendrons, the
formation of cross-linked hydrogels, the analysis of hydrogel
physical properties, and the use of these biomaterials to seal a
corneal incision: the wound created during a typical cataract
procedure.
Figure 1. Structure of macromer 5 and the thiazolidine linkage formed by
the reaction of 3 or 4 and 5. Schematic of an idealized cross-linked hydrogel
formed between 3 and 5.
Scheme 1. Synthetic Route to Peptide Dendrons 3 and 4a
To afford a hydrogel under mild aqueous conditions while
maintaining chemoselective cross-linking and a high tolerance to
a range of chemical functionalities, we selected the formation of a
thiazolidine linkage between the macromers to render a cross-linked
hydrogel (Figure 1). This linkage between an aldehyde and a
cysteine 1,2 aminothiol group occurs quickly at room temperature
and is stable from pH ≈ 3-9. Moreover, this coupling reaction
belongs to a family of peptide ligation reactions used to prepare
large proteins and enzymes in aqueous solution that were otherwise
unattainable using solid-phase peptide synthetic methods.15-17 To
use such a strategy for hydrogel formation, lysine-based peptide
dendrons18,19 possessing either four or eight N-terminal cysteine
residues were synthesized (Scheme 1).
The pentafluoro-ester strategy was used to synthesize the
dendrons since it afforded higher amide coupling reaction yields
than coupling reactions employing BOP, DCC, EDCI, or oxalyl
chloride. First, the pentafluoroesters of ZLys(Z)OH and IsoCys-
(Boc)OH were prepared using DCC and 2,3,4,5,6-pentafluorophenol
(PFP). Next, ZLys(Z)OPFP was coupled to LysOMe‚2HCl in the
a Reactants and conditions: (a) Z-Lys(Z)-OPFP, DMF, DIEA, HOBT,
CH2Cl2, 25 °C for 24 h, 98% yield; (b) H2/Pd/C, MeOH, 6 h, 99% yield;
(c) HCl 1 N, 25 °C for 5 min, 99% yield; (d) iso-Cys(Boc)-OPFP, DMF,
CH2Cl2, DIEA, HOBT, 25 °C for 24 h, 96% yield; (e) TFA 17% in CH2Cl2,
25 °C for 2 h, 97% yield; (f) HCl 1 N/MeOH 50/50, 25 °C for 4 h, 92%
yield; (g) Boc-Lys(Boc)-OPFP, DMF, CH2Cl2, DIEA, HOBT, 25 °C for
24 h, 65% yield; (h) TFA 15% in CH2Cl2, 25 °C for 1 h, 99% yield; (i)
iso-Cys(Boc)-OPFP, DMF, CH2Cl2, DIEA, HOBT, 25 °C for 24 h, 96%
yield; (j) TFA 17% in CH2Cl2, 25 °C for 2 h, 97% yield; (k) HCl 1 N/MeOH
50/50, 25 °C for 4 h, 99% yield.
presence of DIEA and HOBT. The Z group was removed via
catalytic hydrogenolysis (Pd/C (10% w/w) and H2). The dendron
3 was prepared by coupling IsoCys(Boc)OPFP to LysLys(Lys)-
OMe‚4HCl followed by removal of the Boc and iso-protecting
groups of cysteine using TFA and 1 N HCl in MeOH, respectively.
† Boston University.
‡ Duke University Medical Center.
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10.1021/ja045870l CCC: $27.50 © 2004 American Chemical Society

C O M M U N I C A T I O N S
2)-treated eyes were 24 ( 8 and 54 ( 16 mmHg, respectively.
The leaking pressure for the eyes repaired with hydrogel sealant
(N ) 8) was 184 ( 79 mmHg. Normal intraocular pressure is
between 12 and 16 mmHg. The incision is not sealed using only
the dendron or PEG-DA hydrogel precursors. The hydrogel sealant
secures the clear corneal incision and withstands higher pressures
and stress placed on a wound than conventional suture or self-
sealed treated wounds. The procedure with the hydrogel sealant is
facile and requires less surgical time than conventional suturing
(4-6 times) and does not inflict additional tissue trauma. The cross-
linked hydrogel sealant is transparent, adhesive, elastic, hydrophilic,
and acts as a physical barrier to the ocular surface.
In summary, a new in situ polymerizing hydrogel is reported
that gels within a few minutes from the multiple thiazolidine
linkages formed between the cysteine residues of the dendron and
the poly(ethylene glycol) macromers. The use of a hydrogel sealant
as opposed to nylon sutures or the self-seal approach in cataract
surgeries may provide a facile method to safely and effectively
seal the incision while reducing potential complications. These
results further support the synthesis and evaluation of dendritic
macromolecules for medical applications, where a high level of
molecular control can be used to vary and optimize chemical,
physical, and mechanical properties.
Figure 2. (left) Photograph of a synthesized 3‚5 hydrogel and (right)
photograph of a 3‚5 hydrogel sealant-repaired 3-mm clear corneal incision
(incision is between the two purple dots and gel is within the blue border).
The larger dendron 4 was prepared in a similar manner except that
an additional Lys coupling reaction was performed. In this reaction,
the BocLys(Boc)OH was used instead of ZLys(Z)OH to increase
the solubility of the larger Lys-dendron intermediate in organic
solvents. Finally, the Boc and iso-protecting groups of cysteine were
removed using TFA and 1 N HCl in MeOH, respectively, to afford
dendron 4. The dendrons were fully characterized by NMR, FAB-
MS, and elemental analysis (see Supporting Information).
To prepare the hydrogels, dendron 3 or 4 was reacted with poly-
(ethylene glycol dialdehyde) of 3400 Mw (5, PEG-DA) in HEPES
buffer at pH ) 7.4 (Figure 1). The ratio of cysteine to aldehyde
was 1:1, and the total concentration of polymer in solution was
either 30 or 50% w/w. A hydophilic gel formed spontaneously
within three minutes upon mixing the two aqueous solutions at
either concentration (Figures 1 and 2). The gels exhibited vis-
coelastic properties. Cylindrical hydrogel samples of 8-mm diameter
and 2-mm thickness were prepared and analyzed after setting at
25 °C for 24 h. The mechanical properties were measured at a
frequency of 1 Hz. The complex modulus (G*) for the 30% w/w
hydrogels prepared from 3 or 4 and 5 was 3.8 × 103 and 1.0 ×
104, respectively. The increase in modulus is consistent with the
increase in cross-linking density present in the 4‚5 hydrogel.20 The
hydrogels were transparent, and the 3‚5 hydrogel had a refractive
index of 1.397 at 37 °C, similar to the human cornea (n37 ) 1.376).
Cataract removal is the most commonly performed ophthalmic
surgical procedure, with 11 million/year performed worldwide, and
this number is expected to increase with the aging demographics.
A cataract is the clouding of the lens that decreases vision. Today,
surgeons break up and remove the cataract using ultrasound energy
and implant a synthetic intraocular lens, all through an incision in
the cornea. At the conclusion of the procedure, this corneal incision
is either left alone to “self-seal” or closed with nylon sutures.
Suturing inflicts trauma to the tissue and has risks such as
increasing inflammation and vascularization.21-23 Moreover, sutures
need to be removed post-operatively. The “self-seal” approach also
has its drawbacks, including leakage and increased risks of infection
(i.e., endophthalmitis).24 To determine whether this hydrogel sealant
would secure a corneal incision, we performed a series of
experiments on enucleated eyes to evaluate the leaking pressures
of self-sealed, suture, or hydrogel sealant-repaired incisions.
A 3-mm clear corneal linear incision was made in an enucleated
eye. This wound was either left to self-seal or closed using one
interrupted 10-0 nylon suture or the hydrogel sealant. For the
hydrogel sealant, dendron 3 and PEG-DA, 5, were mixed quickly
at room temperature, and then ∼5 µL of the hydrogel sealant was
applied to the incision (Figure 2). Within 5 min of repairing the
incision, saline was injected in the anterior chamber via a syringe
pump until the repaired incision leaked. In this in vitro study, the
mean leaking pressure for the self-seal (N ) 7) and suture (N )
Acknowledgment. This work was supported by the NIH and the
Johnson and Johnson Focused Giving Program. We thank the North
Carolina Eye Bank. M.W.G. also thanks the Dreyfus Foundation for a
Camille Dreyfus Teacher-Scholar Award and the Alfred P. Sloan
Foundation for a Research Fellowship.
Supporting Information Available: Experimental details. This
material is available free of charge via the Internet at http://pubs.acs.org.
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