Osteotropic Peptide That Differentiates Functional Domains of the Skeleton
Dong Wang,†,‡,⊥Scott C. Miller,§,⊥Luda S. Shlyakhtenko,†Alexander M. Portillo,†Xin-Ming Liu,†
Kongnara Papangkorn,‡Pavla Kopec ˇkova ´,‡Yuri Lyubchenko,†William I. Higuchi,‡and Jindr ˇich Kopec ˇek*,‡,|
Department of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical Center, Omaha, Nebraska
68198-6025, Department of Pharmaceutics and Pharmaceutical Chemistry and Department of Bioengineering, University of
Utah, Salt Lake City, Utah 84112, Department of Radiology/Radiobiology Division, University of Utah, Salt Lake City,
Utah 84108.Received June 14, 2007; Revised Manuscript Received July 27, 2007
HPMA copolymer-D-aspartic acid octapeptide (D-Asp8) conjugates have been found to target the entire skeleton
after systemic administration. In a recent study using the ovariectomized rat model of osteoporosis, we surprisingly
discovered that D-Asp8would favorably recognize resorption sites in skeletal tissues, while another bone-targeting
moiety, alendronate (ALN), directs the delivery system to both formation and resorption sites. Atomic force
microscopy (AFM) analyses reveal that ALN has a stronger binding force to hydroxyapatite (HA) than D-Asp8.
In Vitro HA binding studies indicate that D-Asp8 is more sensitive to change of HA crystallinity than ALN.
Because the bone apatite in the newly formed bone (formation sites) usually has lower crystallinity than the
resorption sites (mainly mature bone), we believe that the favorable recognition of D-Asp8to the bone resorption
sites could be attributed to its relatively weak binding to apatite, when compared to bisphosphonates, and the
different levels of crystallinity of bone apatite at different functional domains of the skeleton.
Bone is a specialized connective tissue, which provides
mechanical support and participates in calcium homeostasis. It
is continuously being resorbed and rebuilt to maintain its normal
function. Disturbances of this resorption/formation balance are
characteristic of most bone diseases (1-3). With ever-increasing
understanding of bone biology, many new therapeutic agents
have been identified for the treatment of bone diseases (4-
15). However, most of them do not have tissue specificity to
the skeleton (osteotropicity), which hampers their clinical
application due to the side effects.
In order to overcome this problem, we have developed bone-
targeting water-soluble polymeric drug delivery systems. In the
initial study, alendronate and D-Asp8were used as the bone-
targeting moieties and conjugated to fluorescein isothiocyanate
(FITC)-labeled N-(2-hydroxypropyl)methacrylamide (HPMA)
copolymers (Figure 1A). The administration of these polymers
to young growing balb/c mice led to their strong deposition to
the skeleton, especially at high turnover sites in bone as
determined histologically (16). A later pharmacokinetics and
biodistribution study confirmed that HPMA copolymer using
D-Asp8 as the bone-targeting moiety could recognize the
skeleton, especially the high bone turnover sites, such as tibia
and femur heads, lumbar vertebrae, and mandibular bone (17).
To investigate whether the delivery system could deliver model
drugs to the osteopenic skeleton, FITC-labeled HPMA copoly-
mer-alendronate conjugate (P-ALN-FITC) and FITC-labeled
HPMA copolymer-D-Asp8conjugate (P-D-Asp8-FITC) were
administered intravenously to ovariectomized (OVX) rats, a
common model of postmenopausal osteoporosis. To allow the
development of osteopenia, the OVX procedure was performed
3 months prior to the beginning of the study. To assist in the
identification of different functional domains on bone surfaces,
tetracycline (a fluorochrome marker of bone formation) (18)
was administered intraperitoneally (20 mg/kg) 3 days prior to
the administration of the conjugates. Twenty-four hours after
administration of the conjugates, the OVX rats (three/group)
were euthanized. Tibias, femurs, fifth lumbar vertebra, and
mandibles were isolated, fixed, and processed for undecalcified
As shown in Figure 1B and C, both conjugates were found
to bind well to some bone surfaces in the OVX rats, which
agrees with a previous study using young balb/c mice (16). The
tetracycline marker labeled bone formation surfaces permitted
easier identification of both bone formation and bone resorption
sites. The presence of osteoclasts on the resorption surface and
osteoblasts on the formation surfaces was confirmed by histol-
ogy. Surprisingly, P-D-Asp8-FITC was found to preferentially
bind to resorption surfaces, while P-ALN-FITC appeared to bind
well at both formation and resorption surfaces. Such a result
has never been reported before. As we examine the zone of
dentine calcification, the binding of the P-ALN-FITC appeared
to be stronger than P-D-Asp8-FITC (Figure 1D and E) in the
continuously erupting incisor in the mandible.
Because of this intriguing observation, we hypothesized that
the binding strength of D-Asp8 to bone apatite (a poorly
crystallized, CO3-containing, Ca-deficient hydroxyapatite ana-
log) (19) may be weaker than that of alendronate. Therefore, it
could be sensitive to the crystallinity of bone apatite. It is known
that certain pathophysiological conditions such as Paget’s
disease (20) and osteoporosis (21) can result in changes of the
size of hydroxyapatite crystals. It is also understood that the
crystallinity of newly formed bone mineral at active bone
* Correspondence should be addressed to Jindr ˇich Kopec ˇek, Depart-
ment of Pharmaceutics and Pharmaceutical Chemistry, University of
Utah, 30 S. 2000 E. Rm. 201, Salt Lake City, Utah 84112-5820, USA;
†Department of Pharmaceutical Sciences, College of Pharmacy,
University of Nebraska Medical Center.
‡Department of Pharmaceutics and Pharmaceutical Chemistry,
University of Utah.
§Department of Radiology/Radiobiology Division, University of
|Department of Bioengineering, University of Utah.
⊥These authors contributed equally to this work.
Bioconjugate Chem. 2007, 18, 1375−1378
10.1021/bc7002132 CCC: $37.00© 2007 American Chemical Society
Published on Web 08/17/2007
formation sites is lower than that at the more mature sites where
resorption occurs (20). This may help to explain the observed
preferential binding of P-D-Asp8-FITC to resorption surfaces
in skeletal tissues.
To test this hypothesis, we employed force spectroscopy to
directly measure the interaction of alendronate and D-Asp8with
hydroxyapatite. Since the AFM force measurement requires a
rather flat substrate, we chose tooth enamel (Figure 2) as a model
of hydroxyapatite surface. Alendronate or D-Asp8 was co-
valently attached to the AFM probes via glutaraldehyde cross-
links (22-24). Force-distance measurements were performed
in PBS buffer at room temperature at identical experimental
conditions. Force curves were taken at various random locations
on the surface in order to account for any heterogeneity on the
surface. Although both modified tips clearly indicated the tip-
surface interaction (Figure 3A and B), the most frequent force-
distance curves were different for alendronate and D-Asp8.The
force distance curves for the D-Asp8-modified tip more often
showed a single adhesion peak, whereas the alendronate-
modified tip often had two clear peaks, including adhesion. Each
individual force plot was analyzed one at a time. The rupture
force and the distance for each peak were also measured. The
results obtained for a series of measurements of adhesion forces
for the tips modified with alendronate and D-Asp8, respectively,
are shown as histograms in Figure 3C and D. Both histograms
demonstrate the interaction of modified tips with the tooth
enamel surface, but they are clearly different. For alendronate,
the distribution of forces is wide with majority of the forces
recorded around 190 pN. For D-Asp8, the distribution is
narrower with the majority of the forces recorded around 105
pN. In addition to the analyses of adhesion forces at zero
distances, secondary peaks at distances away from the adhesion
peak were also analyzed and shown in Figure 3E and F. Clearly,
the data reveal a more pronounced difference in the interaction
patterns for alendronate and D-Asp8. First, the number of
secondary events was considerably larger for alendronate
compared to D-Asp8. Second, the values for rupture forces were
higher for alendronate than for D-Asp8. An additional difference
was the position of the secondary peak. This effect is graphically
illustrated in Figure 3G and H, on which the histograms for the
distance distribution are plotted for alendronate and D-Asp8,
respectively. The positions for the secondary rupture events are
centered around 40 nm for alendronate, whereas the secondary
peaks for D-Asp8 are scattered but with the majority of the
events at distances of ∼10 nm. In spite of the heterogeneity in
chemical composition and roughness of the enamel surface
(Figure 2), which may widen the histograms for the interaction
forces and distances, these AFM data strongly suggest that
alendronate interacts more efficiently with the tooth enamel
surface and has less sensitivity to the surface topography in
comparison with D-Asp8.
To further test the possibility that alendronate and D-Asp8
will bind differently to hydroxyapatite with different crystal-
linity, we synthesized two hydroxyapatites (HAa, high crystal-
linity, and HAb, low crystallinity; Table 1) (25, 26) for the in
Vitro binding test. Solubility studies and X-ray diffraction
evaluation of these hydroxyapatites indicate that the lattice
disorder of the bulk mineral phase (which is reflected by the
crystallite microstrain parameter value) correlates well with the
lattice disorder at the crystal-solution interface (to be published).
So, it would be reasonable to expect that the binding behavior
of molecules to apatite surfaces can be dependent upon the
lattice disorder of the bulk mineral phase. PBS solutions of
P-ALN-FITC and P-D-Asp8-FITC were incubated with HAaand
HAb. After removal of HA powders from the solutions, the
percentage of the FITC-labeled conjugates bound to HAs was
calculated from the decreased UV absorbance of the polymer
solutions. As shown in Figure 4, the higher absolute binding
Figure 1. Different in ViVo bone-binding abilities of HPMA copoly-
mers conjugated with alendronate (P-ALN-FITC) and D-aspartic acid
octapeptide (P-D-Asp8-FITC). (A) Chemical structures of the osteotropic
polymer conjugates. (B) Cancellous bone from an ovariectomized
(OVX) rat injected with the bone formation marker tetracycline (TC)
and the P-ALN-FITC. Both tetracycline (TC, yellow) and P-ALN-FITC
(green) are seen on some surfaces (arrows) indicating uptake on bone-
forming surfaces. A selected area is magnified (×2, insert) to show
the double label of TC and P-ALN-FITC. P-ALN-FITC is also evident
on some resorption surfaces (double arrows). Bar ) 100 µm. (C)
Cancellous bone from an OVX rat injected with TC and the P-D-Asp8-
FITC. Uptake of TC but little of P-D-Asp8-FITC is evident on bone
formation surfaces (arrows). P-D-Asp8-FITC is evident on bone
resorption surfaces (double arrows). Bar ) 100 µm. (D) Cross sections
of the forming mandibular incisor illustrating the uptake of the P-ALN-
FITC. Bar ) 50 µm. (E) Cross sections of the forming mandibular
incisor illustrating the uptake of the P-D-Asp8-FITC. Bar ) 50 µm.
The TC label (yellow) is clearly evident in both sections (arrows). There
is more apparent uptake of P-ALN-FITC (green label, double arrows)
in the mineralizing dentin than uptake of P-D-Asp8-FITC.
Figure 2. AFM amplitude image of tooth enamel surface taken in
PBS buffer using nonmodified tip. Bar ) 2 µm.
Bioconjugate Chem., Vol. 18, No. 5, 2007Wang et al.
percentage of P-ALN-FITC to HAs, when compared to P-D-
Asp8-FITC, is probably confirming the stronger binding force
of alendronate than D-Asp8. Nevertheless, the binding of
P-ALN-FITC to HAs is less sensitive to the change of apatite
crystallinity compared to P-D-Asp8-FITC.
Evidently, the AFM study suggests that alendronate has
stronger binding to hydroxyapatite than D-Asp8. Such differ-
ences in their ability to bind to hydroxyapatite coupled with
their differences in binding with changes in apatite crystallinity
would provide an explanation for the preferential binding of
D-Asp8to hydroxyapatite with higher crystallinity. Though these
experiments were not performed on bone apatite, the data
support the hypothesis that it is the weak binding ability of
D-Asp8to apatite that causes its in ViVo selectivity to the bone
resorption surface (containing bone apatite with relatively higher
crystallinity) over formation surfaces (mainly amorphous cal-
cium phosphate) (19). Incorporation of molecular structures
(e.g., tetracycline, D-Asp8) that could recognize different sub-
tissue functional domains (osteolytic and osteoblastic) (18) into
the new drug design and osteotropic drug delivery systems
would greatly enhance the therapeutic index by delivering the
drug directly to the desired cells and molecular targets.
We acknowledge financial support from the College of
Pharmacy, University of Nebraska Medical Center (D.W.,
L.S.S., A.M.P., X.M.L., and Y.L.) and NIH Grant GM069847
(S.C.M., P.K., and J.K.).
Supporting Information Available: Detailed AFM procedures
are described in the Supporting Information. This material is
available free of charge via the Internet at http://pubs.acs.org.
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Table 1. Characterization of Synthetic Hydroxyapatite with
aThe remaining contents in HAaand Habare mainly water, hydroxide,
and trace elements (27).
Figure 4. Binding ability of P-ALN-FITC and P-D-Asp8-FITC to
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