US2022313609  --  Nano composite material aiming at acidic sealing zone in osteoclasts and preparation method thereof

Extracted document text. Layout, formulas and symbols may not survive extraction; consult the preserved PDF for the original presentation.

Text extraction covers 11 pages, including 11 transcribed by optical character recognition.

Page 1

as) United States

US 202203

13609A1

a2) Patent Application Publication co) Pub. No.: US 2022/0313609 Al

Lin et al. (43) Pub. Date: Oct. 6, 2022
(54) NANO COMPOSITE MATERIAL AIMING AT AGIK 47/10 (2006.01)
ACIDIC SEALING ZONE IN OSTEOCLASTS AGIK 31/65 (2006.01)
AND PREPARATION METHOD THEREOF AGIK 33/00 (2006.01)
AGIP 19/10 (2006.01)
(71) Applicant: SIR RUN RUN SHAW HOSPITAL (52) US. Ch.
ZHEJIANG UNIVERSITY SCHOOL CPC veces, AGIK 9/1273 (2013.01); AGIK 47/24

OF MEDICINE, Hangzhou, Zhejiang
(CN)

Xianfeng Lin, Hangzhou, Zhejiang
(CN); Chenhui Gu, Hangzhou,
Zhejiang (CN); Qingqing Wang,
Hangzhou, Zhejiang (CN); Shunwu
Fan, Hangzhou, Zhejiang (CN)

(72) Inventors:

(21)
(22)

Appl. No. 17/426,535

PCT Filed: Apr. 10, 2020

(86) PCT No.:

§ 371 (©),
(2) Date:

PCT/CN2020/084300

Jul. 28, 2021
Publication Classification

Int. Cl.
AG6IK 9/127
AGIK 47/24

(51)
(2006.01)
(2006.01)

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(2013.01); AGIK 47/10 (2013.01); AGIK 31/65
(2013.01); A61K 33/00 (2013.01); A6IP 19/10
(2018.01)

(57) ABSTRACT

A nano composite material aiming at an acidic sealing zone
in osteoclasts and a preparation method thereof are provided.
The nano composite material aiming at the acidic sealing
zone in the osteoclasts includes a nano material, bone-
targeting molecules, and a compound able to react with the
acidic sealing zone in the osteoclasts, wherein: after being
modified by the bone-targeting molecules, the nano material
is loaded with the compound able to react with the acidic
sealing zone in the osteoclasts. Through accurate mature
osteoclast targeting and chemically regulated biocascade
effects, the osteoclasts are inhibited, which provides a new
idea and a new tool for drug therapy of abnormal osteoclast
activation.

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Page 2

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Page 4

Patent Application Publication Oct. 6,2022 Sheet 3 of 4 US 2022/0313609 Al

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Patent Application Publication | Oct. 6,2022 Sheet 4 of 4 US 2022/0313609 Al

in vivo study.

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Page 6

US 2022/0313609 Al

NANO COMPOSITE MATERIAL AIMING AT
ACIDIC SEALING ZONE IN OSTEOCLASTS
AND PREPARATION METHOD THEREOF

BACKGROUND OF THE PRESENT
INVENTION

Field of Invention

[0001] The present invention relates to a field of drug
therapy for bone tissues, and more particularly to a nano
composite material aiming at an acidic sealing zone in
osteoclasts and a preparation method thereof.

Description of Related Arts

[0002] Osteoporosis is a global chronic disease character-
ized by severe bone loss and bone fracture, which brings
pains to the patients and greatly reduces the life quality. The
bone tumor and the abnormal osteoclast activation during
the bone metastasis of the tumor can also cause the abnormal
osteoclast activation, thereby causing osteoporosis and
pathological bone fracture. The conventional treatment
means for the abnormal osteoclast activation comprise the
bone resorption inhibitors such as the calcium preparation,
vitamin D, calcitonin, bisphosphonate and estrogen, and the
bone formation promoters such as the fluoride, anabolic
steroid, and parathyroid hormone. Although the above treat-
ment means can affect the function of the osteoclasts or
facilitate bone formation to slow down the disease progres-
sion, the bone loss cannot be completely inhibited as the
acidification and bone destructions caused by the osteoclasts
are irreversible. The acidification of the contact interface of
the osteoclasts with the bones is the root cause of dissolution
and organic degradation of the bone minerals during osteo-
porosis. Thus, it is urgent to develop a material aiming at the
acidic sealing zone in the osteoclasts, so as to prevent and
treat the abnormal osteoclast activation.

[0003] There are numerous nano materials can be used in
drug therapy of the bone tissues, such as the liposomes,
polymer nano particles, silicon dioxide particles and nano
coatings. Through the specific targeting way and drug
release way, these nano materials can achieve the therapeutic
effect. When treating osteoporosis, these nano materials can
reach the targeted bone tissues and affect the function of the
osteoclasts in a certain way. However, the common materials
have the problems of inaccurate targeting, insignificant
effect, and great toxic and side effects. The Chinese patent
application of CN 201710283530.5 disclosed a preparation
method of a dual-targeting drug-loaded nano-particle lipid-
polymer for osteoporosis, which enhances the targeting
effect of the drug to decrease the side effects of the drug. The
Chinese patent application of CN 201710841290.6 disclosed
an application of a pH-responsive nano material in prepa-
ration of a bone resorption inhibitor for preventing and
treating osteoporosis, which selectively inhibits the osteo-
clasts with the pH-responsive graphene oxide, chitosan or
hydrogel. The above patent applications optimize the deliv-
ery or release process to a certain extent.

[0004] However, it is difficult for the conventional mate-
rials to solve the two problems at the same time, namely
accuracy of osteoclast targeting and physiology safety of the
drug. During the progression of osteoporosis, the bone
destructions caused by bone resorption of the mature osteo-
clasts as well as the bone repair and homeostasis maintain-

Oct. 6, 2022

ing mediated by other cells are important. Thus, the rational
materials for treating osteoporosis should be able to target
the bone tissues, especially the acidified osteoclasts, in the
circulation. Meanwhile, the materials used for targeting and
pH response should be materials widely applied in clinic or
common materials in the body. If the above two require-
ments are met, inhibition on the osteoclasts can be realized,
while toxic and side effects on other cells can be as small as
possible, so as to achieve better resistance to bone resorption
and better facilitation on bone formation.

SUMMARY OF THE PRESENT INVENTION

[0005] Aiming at deficiencies in prior art, the present
invention provides a nano composite material aiming at an
acidic sealing zone in osteoclasts and a preparation method
thereof. Through bone targeting with a common clinical
drug, accurate targeting and function inhibition of the osteo-
clasts are both realized by a chemical reaction.

[0006] Inorder to accomplish the above object, the present
invention adopts technical solutions as follows.

[0007] A nano composite material aiming at an acidic
sealing zone in osteoclasts comprises a nano material, bone-
targeting molecules, and a compound able to react with the
acidic sealing zone in the osteoclasts, wherein: after being
modified by the bone-targeting molecules, the nano material
is loaded with the compound able to react with the acidic
sealing zone in the osteoclasts; the nano material is loadable
and modifiable; the bone-targeting molecules have an obvi-
ous affinity to bone tissues; and the compound able to react
with the acidic sealing zone in the osteoclasts is alkalescent
or neutral bicarbonate.

[0008] Preferably, the nano material is liposomes, polymer
nano particles or mesoporous silicon oxide particles.
[0009] Preferably, the bone-targeting molecules are tetra-
cycline, phosphonate or aspartic acid polypeptide
sequences.

[0010] Preferably, the compound able to react with the
acidic sealing zone in the osteoclasts is sodium bicarbonate,
potassium bicarbonate or ammonium bicarbonate; further
preferably, the compound is sodium bicarbonate having a
concentration of | mol/L.

[0011] A method for preparing the nano composite mate-
rial aiming at the acidic sealing zone in the osteoclasts
comprises steps of: cross-linking the loadable and modifi-
able nano material with the bone-targeting molecules: dis-
solving in chloroform with lecithin and cholesterol, and
controlling a pH value to 8.0-8.4; at a room temperature,
magnetically stirring and cross-linking for 24-72 hours;
forming a membrane in a rotary evaporator, adding a
solution to be loaded, and shaking for hydration; ultrasoni-
cally emulsifying, and dialyzing; wherein: a molar ratio of
the loadable and modifiable nano material to the bone-
targeting molecules is 1:1-1:2.

[0012] Preferably, a functionalized molecule in the load-
able and modifiable nano material is functionalized phos-
pholipid; after cross-linking with the bone-targeting mol-
ecules, bone-targeting functionalized phospholipid is
obtained.

[0013] Preferably, the step of “ultrasonically emulsifying”
specifically comprises steps of: turning on for 1-2 seconds
with a power of 30-70%, then turning off for 2-3 seconds,
and repeating for 5-20 minutes; and the step of “dialyzing”
lasts for 1-3 days.

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US 2022/0313609 Al

[0014] Preferably, the functionalized phospholipid is
DSPE-PEG-NHS; and the bone-targeting molecules are
tetracycline, namely TC.

[0015] The present invention has beneficial effects as
follows. The present invention provides the nano composite
material aiming at the acidic sealing zone in the osteoclasts
and the preparation method thereof. Through accurate
mature osteoclast targeting and chemically regulated bio-
cascade effects, the osteoclasts are inhibited, which provides
a new idea and a new tool for drug therapy of abnormal
osteoclast activation.

[0016] Compared with the conventional drug or material
for treating osteoporosis, the present invention achieves
significant progresses as follows.

[0017] 1) Accurate dual-targeting is conducted with the
bone tissue-targeting molecules and the pH response aiming
at the acidic sealing zone in the osteoclasts, so that the
utilization of the drug is improved and the side effects on
other tissues and organs are reduced.

[0018] 2) The used drug is the physiological compound
existing in the human body, with the low toxicity. It can
serve as the component generating the therapeutic effect and
the component of rapid pH response at the same time, so that
dual functions are achieved.

[0019] 3) The resistance to osteoclastic bone erosion is
verified by the in-vitro experiment. The present invention
has the obvious inhibitory effects on the count and area of
the osteoclasts, the resorbed count and the resorbed area.
[0020] 4) The facilitation on formation of the exosomes by
the osteoclasts is verified by the in-vitro experiment. With
utilizing the receptor activator of nuclear factor-kappa B
(RANK) on the surface of the exosomes, ineffective binding
with the receptor activator of nuclear factor-kappa B ligand
(RANKL) in serum is realized, thereby realizing the long-
term inhibitory effects on the osteoclasts.

[0021] 5) The resistance to osteoporosis is verified by the
in-vivo experiment. The present invention has the significant
improvements on bone volume per tissue volume, trabecular
number, and trabecular separation.

[0022] 6) As a model of the nano composite material
aiming at the acidic sealing zone in the osteoclasts, each
component is replaceable, which has the great referential
significance.

[0023] In conclusion, the nano composite material aiming
at the acidic sealing zone in the osteoclasts, provided by the
present invention, can be applied in preventing and treating
the abnormal activation of the osteoclasts, showing the
obvious therapeutic effect.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The patent of application file contains at least one
drawing executed in color. Copies of this patent or patent
application publication with color drawing(s) will be pro-
vided by the Office upon request and payment of the
necessary fee.

[0025] In order to more clearly illustrate objects, technical
solutions and beneficial effects of the present invention, the
accompanying drawings are described as follows.

[0026] FIG. 1(a)-FIG. 1(g) show preparation and repre-
sentation of a nano composite material aiming at an acidic
sealing zone in osteoclasts according to the present inven-
tion, wherein: FIG. 1(@) is a sketch view of an action
mechanism of the nano composite material aiming at the
acidic sealing zone in the osteoclasts; FIG. 1(6) and FIG.

Oct. 6, 2022

1(c) are sketch views of a preparation process of the nano
composite material aiming at the acidic sealing zone in the
osteoclasts; FIG. 1(d)-FIG. 1(/) show verification of cross-
linking between bone-targeting molecules and functional-
ized phospholipid respectively through a matrix-assisted
laser desorption/ionization time of flight (MALDI-TOF)
mass spectrometer, a laser scanning confocal microscope
and a fluorescent spectrophotometer; and FIG. 1(g) shows
representation of the nano composite material aiming at the
acidic sealing zone in the osteoclasts under a cryo-transmis-
sion electron microscope.

[0027] FIG. 2(@)-FIG. 2(i) show verification of functions
of the nano composite material aiming at the acidic sealing
zone in the osteoclasts according to the present invention,
wherein: FIG. 2(a) shows verification of an acid resistance
of the nano composite material aiming at the acidic sealing
zone in the osteoclasts by an acidometric titration experi-
ment; FIG. 2(5) shows verification of a pH response of the
nano composite material aiming at the acidic sealing zone in
the osteoclasts by measuring a particle size under different
pH values; FIG. 2(c) and FIG. 2(@) show verification of
mechanical changes of the nano composite material aiming
at the acidic sealing zone in the osteoclasts under different
pH values through an in-situ liquid atomic force microscope;
FIG. 2(e) show verification of a release of the nano com-
posite material aiming at the acidic sealing zone in the
osteoclasts under an acidic condition (pH=4) through the
cryo-transmission electron microscope; FIG. 2(/) and FIG.
2(g) show verification of long-term (7 days) stability and
rapid pH response of the nano composite material aiming at
the acidic sealing zone in the osteoclasts through an in-vitro
fluorescence microscope; FIG. 2(h) show verification of a
rapid enrichment effect of the nano composite material
aiming at the acidic sealing zone in the osteoclasts in
targeted bones of mice through in-vivo fluorescence; and
FIG. 2(i) show verification of an inhibitory effect of the nano
composite material aiming at the acidic sealing zone in the
osteoclasts on osteoclastic bone erosion through a fluores-
cence confocal microscope.

[0028] FIG. 3(a)-FIG. 3(4) show inhibitory effects of the
nano composite material aiming at the acidic sealing zone on
the osteoclasts through chemically regulated biocascade
effects according to the present invention, wherein: FIG.
3(a) show verification of an inhibitory effect of the nano
composite material aiming at the acidic sealing zone on the
osteoclasts through tartrate resistant acid phosphatase
(TRAP)-staining; FIG. 3(b) show verification of a signifi-
cant improvement of the nano composite material aiming at
the acidic sealing zone in the osteoclasts on the osteoclastic
bone erosion through the scanning electron microscope;
FIG. 3(c) and FIG. 3(d) show verification of inhibition of the
nano composite material aiming at the acidic sealing zone in
the osteoclasts on an increment effect of NFATc-1, c-Fos and
CTSK expressions in the osteoclasts with time through
Western-blot and quantitative polymerase chain reaction
(q-PCR); FIG. 3(e) show verification of an inhibitory effect
of the nano composite material aiming at the acidic sealing
zone in the osteoclasts on formation of the acidic sealing
zone in the osteoclasts through the laser scanning confocal
microscope; FIG. 3(f) and FIG. 3(g) show verification of
inhibition of the nano composite material aiming at the
acidic sealing zone in the osteoclasts on an increment effect
of receptor activator of nuclear factor-kappa B (RANK)
expressions in the osteoclasts with time through Western-

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US 2022/0313609 Al

blot and the fluorescence confocal microscope; FIG. 3(h)
and FIG. 3(/) show verification of facilitation of the nano
composite material aiming at the acidic sealing zone in the
osteoclasts on formation of RANK-containing exosomes by
the osteoclasts through Western-blot and extracellular
vesicle flow cytometry, FIG. 3(7) and FIG. 3(4) show
verification of an inhibitory effect of RANK-containing
extracellular vesicles secreted by the osteoclasts on forma-
tion of the osteoclasts through TRAP staining, wherein
secretion of the RANK-containing extracellular vesicles is
facilitated by the nano composite material aiming at the
acidic sealing zone in the osteoclasts.

[0029] FIG. 4(@)-FIG. 4(/) show inhibitory effects of the
nano composite material aiming at the acidic sealing zone in
the osteoclasts on osteoporosis of ovariectomy (OVX) mice
according to the present invention, wherein: FIG. 4(a) shows
establishment of an animal model, and grouping and evalu-
ation ways; FIG. 4(b) and FIG. 4(c) show verification of
significant improvements of the nano composite material
aiming at the acidic sealing zone in the osteoclasts on bone
volume per tissue volume, trabecular number, and trabecular
separation of vertebral body, femur and tibia of the OVX
mice through micro-CT;, FIG, 4(d) and FIG. 4(e) show
verification of significant improvements of the nano com-
posite material aiming at the acidic sealing zone in the
osteoclasts on bone volume per tissue volume, number of the
osteoclasts, and surface area of the osteoclasts of bone
tissues of the OVX mice through hematoxylin-eosin (H&E)
staining and TRAP staining; and FIG, 4(/) show verification
of a significant inhibitory effect of the nano composite
material aiming at the acidic sealing zone in the osteoclasts
on osteoclast metabolic indicators of the OVX mice through
serological indicators.

DETAILED DESCRIPTION OF THE
PREFERRED EMBODIMENT

[0030] The present invention provides a nano composite
material aiming at an acidic sealing zone in osteoclasts and
a preparation method thereof. The present invention will be
described in detail with the following examples, but these
examples cannot be understood as the limitation to the
protection scope of the present invention.

[0031] A sketch view of an action mechanism of the nano
composite material aiming at the acidic sealing zone in the
osteoclasts is shown in FIG. 1(a).

[0032] According to the present invention, the nano com-
posite material aiming at the acidic sealing zone in the
osteoclasts is sodium bicarbonate-loaded and tetracycline-
modified nano liposomes (NaHCO,-TNLs for short), and
prepared through steps of: dissolving 20.00 mg DSPE-PEG-
NHS and 3.05 mg tetracycline in 10.00 mL chloroform;
adding triethylamine, and regulating a pH value to 8.2; at a
room temperature, magnetically stirring and cross-linking
for 48 hours, and then obtaining DSPE-PEG-TC; dissolving
DSPE-PEG-TC in chloroform with 80.00-120.00 mg leci-
thin and 12.00-20.00 mg cholesterol; forming a membrane
in a rotary evaporator, adding 10 mL sodium bicarbonate
solution having a concentration of 1 mol/L, and shaking for
hydration; ultrasonically emulsifying, specifically compris-
ing steps of turning on for 2 seconds with a power of 40%,
then turning off for 3 seconds, and repeating for 10 minutes;
dialyzing in a dialysis bag for 72 hours; taking out, and

Oct. 6, 2022

filtering with a filter head of 0.22 jum; and preserving at 4°
C. The prepared material is shown in FIG. 1(4) and FIG.
1c).

[0033] The present invention is able to prepare other nano
composite materials aiming at the acidic sealing zone in the
osteoclasts, such as the nano liposomes loaded with ammo-
nium bicarbonate or potassium bicarbonate and modified by
tetracycline or alendronic acid, which can achieve same
technical effects.

EXAMPLE 1

Preparation of sodium bicarbonate-loaded and
tetracycline-modified Nano Liposomes

[0034] The preparation process comprises steps of:

[0035] (1) dissolving 20.00 mg DSPE-PEG-NHS and 3.05
mg tetracycline in 10.00 mL chloroform; adding triethylam-
ine, and regulating a pH value to 8.2; at a room temperature,
magnetically stirring and cross-linking for 48 hours;

[0036] (2) dissolving a product obtained in the step (1) in
chloroform with 100.00 mg lecithin and 16.00 mg choles-
terol; and forming a membrane in a rotary evaporator;

[0037] (3) adding 10 mL sodium bicarbonate solution
having a concentration of 1 mol/L into a flask, and shaking,
for hydration;

[0038] (4) ultrasonically emulsifying, specifically com-
prising steps of: turning on for 2 seconds with a power of
40%, then turning off for 3 seconds, and repeating for 10
minutes; and

[0039] (5) dialyzing in a dialysis bag for 72 hours; taking
out, and filtering with a filter head of 0.22 jum; and preserv-
ing at 4° C.

EXAMPLE 2

Preparation of sodium bicarbonate-loaded and
alendronic acid-modified Nano Liposomes

[0040] The preparation process comprises steps of:

[0041] (1) dissolving 20.00 mg DSPE-PEG-NHS and 2.30
mg alendronate sodium in 10.00 mL chloroform; adding
triethylamine, and regulating a pH value to 8.2; at a room
temperature, magnetically stirring and cross-linking for 48
hours;

[0042] (2) dissolving a product obtained in the step (1) in
chloroform with 100.00 mg lecithin and 16.00 mg choles-
terol; and forming a membrane in a rotary evaporator;

[0043] (3) adding 10 mL sodium bicarbonate solution
having a concentration of 1 mol/L into a flask, and shaking
for hydration;

[0044] (4) ultrasonically emulsifying, specifically com-
prising steps of: turning on for 2 seconds with a power of
40%, then turning off for 3 seconds, and repeating for 20
minutes; and

[0045] (5) dialyzing in a dialysis bag for 72 hours; taking
out, and filtering with a filter head of 0.22 jum; and preserv-
ing at 4° C.

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US 2022/0313609 Al

EXAMPLE 3

Preparation of potassium bicarbonate-loaded and
tetracycline-modified Nano Liposomes

[0046] The preparation process comprises steps of:
[0047] (1) dissolving 20.00 mg DSPE-PEG-NHS and 3.05
mg tetracycline in 10.00 mL. chloroform; adding triethylam-
ine, and regulating a pH value to 8.2; at a room temperature,
magnetically stirring and cross-linking for 24 hours;
[0048] (2) dissolving a product obtained in the step (1) in
chloroform with 80.00 mg lecithin and 16.00 mg choles-
terol; and forming a membrane in a rotary evaporator;
[0049] (3) adding 10 mL potassium bicarbonate solution
having a concentration of 1 mol/L into a flask, and shaking
for hydration;

[0050] (4) ultrasonically emulsifying, specifically com-
prising steps of: turning on for 2 seconds with a power of
40%, then turning off for 3 seconds, and repeating for 10
minutes; and

[0051] (5) dialyzing in a dialysis bag for 72 hours; taking
out, and filtering with a filter head of 0.22 \um; and preserv-
ing at 4° C.

EXAMPLE 4

Preparation of potassium bicarbonate-loaded and
alendronic acid-modified Nano Liposomes

[0052] The preparation process comprises steps of:
[0053] (1) dissolving 20.00 mg DSPE-PEG-NHS and 2.30
mg alendronate sodium in 10.00 mL chloroform; adding
triethylamine, and regulating a pH value to 8.2; at a room
temperature, magnetically stirring and cross-linking for 48
hours;

[0054] (2) dissolving a product obtained in the step (1) in
chloroform with 120.00 mg lecithin and 16.00 mg choles-
terol; and forming a membrane in a rotary evaporator;
[0055] (3) adding 10 mL potassium bicarbonate solution
having a concentration of 1 mol/L into a flask, and shaking
for hydration;

[0056] (4) ultrasonically emulsifying, specifically com-
prising steps of: turning on for 2 seconds with a power of
40%, then turning off for 3 seconds, and repeating for 5
minutes; and

[0057] (5) dialyzing in a dialysis bag for 72 hours; taking
out, and filtering with a filter head of 0.22 tum; and preserv-
ing at 4° C.

EXAMPLE 5

Preparation of ammonium bicarbonate-loaded and
tetracycline-modified Nano Liposomes

[0058] The preparation process comprises steps of:
[0059] (1) dissolving 20.00 mg DSPE-PEG-NHS and 3.05
mg tetracycline in 10.00 mL chloroform; adding triethylam-
ine, and regulating a pH value to 8.2; at a room temperature,
magnetically stirring and cross-linking for 48 hours:
[0060] (2) dissolving a product obtained in the step (1) in
chloroform with 100.00 mg lecithin and 12.00 mg choles-
terol; and forming a membrane in a rotary evaporator;
[0061] (3) adding 10 mL ammonium bicarbonate solution
having a concentration of 1 mol/L into a flask, and shaking
for hydration;

Oct. 6, 2022

[0062] (4) ultrasonically emulsifying, specifically com-
prising steps of: turning on for 1 second with a power of
40%, then turning off for 3 seconds, and repeating for 10
minutes; and

[0063] (5) dialyzing in a dialysis bag for 24 hours; taking
out, and filtering with a filter head of 0.22 jum; and preserv-
ing at 4° C.

EXAMPLE 6

Preparation of ammonium bicarbonate-loaded and
alendronic acid-modified Nano Liposomes

[0064] The preparation process comprises steps of:
[0065] (1) dissolving 20.00 mg DSPE-PEG-NHS and 2.30
mg alendronate sodium in 10.00 mL chloroform; adding
triethylamine, and regulating a pH value to 8.2; at a room
temperature, magnetically stirring and cross-linking for 48
hours;

[0066] (2) dissolving a product obtained in the step (1) in
chloroform with 100.00 mg lecithin and 20.00 mg choles-
terol; and forming a membrane in a rotary evaporator;
[0067] (3) adding 10 mL ammonium bicarbonate solution
having a concentration of 1 mol/L into a flask, and shaking
for hydration;

[0068] (4) ultrasonically emulsifying, specifically com-
prising steps of turning on for 2 seconds with a power of
70%, then turning off for 2 seconds, and repeating for 10
minutes; and

[0069] (5) dialyzing in a dialysis bag for 72 hours; taking
out, and filtering with a filter head of 0.22 jum; and preserv-
ing at 4° C.

[0070] Composite Evaluation of NaHCO ,-TNLs in
Example 1

[0071] 1. Through respectively detecting mass spectrums
of DSPE-PEG-NHS and DSPE-PEG-TC with the matrix-
assisted laser desorption/ionization time of flight (MALDI-
TOF) mass spectrometer, it is obtained that: a molecular
weight of DSPE-PEG-NHS is distributed at 2900 and a
molecular weight of DSPE-PEG-TC is distributed at 3250,
which are consistent with theoretical molecular weights
thereof (as shown in FIG. 1(@)).

[0072] 2. Fluorescein isothiocyanate (FITC) and sodium
bicarbonate solution are co-loaded; through a laser scanning
confocal microscope, it can be seen that localization of the
tetracycline fluorescence and the liposome membrane are
consistent (as shown in FIG. 1(e)).

[0073] 3. The fluorescent spectrophotometer shows that
the fluorescence intensity of NaHCO;-TNLs is obviously
increased at $25 nm in comparison to NaHCO,-NLs without
tetracycline modification (as shown in FIG. 1(/).

[0074] 4. The cryo-transmission electron microscope
shows that the particle size of NaHCO,-TNLs is nano-scaled
and morphology of NaHCO,-TNLs is uniform.

[0075] Characteristic Evaluation of NaHCO,-TNLs in
Example 1

[0076] 1. A titration experiment is conducted on the
sodium bicarbonate-loaded and tetracycline-modified nano
liposomes (NaHCO,-TNLs), the sodium chloride-loaded
and tetracycline-modified nano liposomes (NaCI-TNLs),
water, the sodium bicarbonate solution having a concentra-
tion of 1 mol/L, and the sodium bicarbonate solution having
a concentration of 0.02 mol/L with 1% hydrochloric acid,
and a dynamic change of the pH value thereof is detected in

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US 2022/0313609 Al

real time. The results show that NaHCO,-TNLs have the
excellent acid resistance (as shown in FIG. 2(a)).

[0077] 2. The particle sizes of NaHCO,-TNLs and NaCl-
TNLs are measured respectively under the pH values of 7,
6 and 4. The results show that the particle size of NaHCO,-
TNLs is obviously decreased in an acid environment of
PH=4, indicating that the contents are released in the acid
environment (as shown in FIG. 2(d)).

[0078] 3. The mechanical characteristics of NaHCO,-
TNLs under the pH values of 7 and 4 are compared through
an in-situ liquid atomic force microscope. The results show
that: the particle size of NaHCO;-TNLs is obviously
decreased under the pH value of 4, and the liposome
membrane tends to rupture (as shown in FIG. 2(c) and FIG.
2@).

[0079] 4. The morphologies of NaHCO,-TNLs under the
pH values of 7 and 4 are compared through the cryo-
transmission electron microscope. The results show that
rupture of the liposome membrane occurs under the pH
value of 4 (as shown in FIG. 2(e)).

[0080] 5. FITC is loaded into NaHICO,-TNLs and there-
after incubated in a 10% serum medium with bovine bone
slices, and the fluorescence respectively 1 day, 3 days and 7
days after incubation is observed; 7 days later, the medium
is placed in an environment with the pH value of 4, and the
liposome fluorescence respectively 0 minute, 1 minute and
3 minutes after placement is observed. The results show that:
NaHCO,-TNLs can be adsorbed on the bone surface in 7
days and keep stable; moreover, NaHCO,-TNLs still have
the rapid pH response function 7 days later.

[0081] 6. Indocyanine green-loaded and tetracycline-
modified nano liposomes (ICG-INLs) and indocyanine
green-loaded nano liposomes without tetracycline modifi-
cation (ICG-NLs) are injected into the caudal veins of mice
with a dosage of 0.025 ml/g. The results show that: com-
pared with ICG-NLs, ICG-TNLs have a significant rapid
enrichment effect on bone tissues (as shown in FIG. 2(A)).
[0082] 7. NaHCO,-TNLs and NaCl-TNLs are respec-
tively co-incubated with the FITC-coated bovine bone
slices, and the mature osteoclasts are respectively implanted
thereon. The results show that the FITC fluorescence inten-
sity and area of the bone surface with NaHCO,-TNLs are
significantly better than that with NaCl-TNLs, indicating
that NaHCO,-TNLs can effectively inhibit the bone erosion
effect of the osteoclasts (as shown in FIG. 2(i)).

[0083] Inhibitory Effects of NaHCO,-TNLs in Example 1
on Osteoclasts

[0084] 1. NaHCO,-TNLs are added into an osteoclast
inducing system and compared with a pure osteoclast induc-
ing system. The results obtained through tartrate resistant
acid phosphatase (TRAP)-staining show that the count and
the area of the osteoclasts are both greatly inhibited with
NaHCO,-TNLs, indicating that NaHCO,-TNLs have the
significant inhibitory effect on the osteoclasts (as shown in
FIG. 3(a)).

[0085] 2. NaHCO,-TNLs are added into an osteoclast
inducing system cultured with the bovine bone slices and
compared with a pure osteoclast inducing system cultured
with the bovine bone slices. The results obtained through the
scanning electron microscope show that an absorption count
and an absorption area are both greatly inhibited with
NaHCO,-TNLs, indicating that NaHCO,-TNLs have the
significant inhibitory effect on the osteoclasts (as shown in
FIG. 3(6)).

Oct. 6, 2022

[0086] 3. NaHCO,-TNLs are added into the osteoclast
inducing system and compared with the pure osteoclast
inducing system. The results obtained through Western-blot
and quantitative polymerase chain reaction (q-PCR) show
that NaHCO,-TNLs can inhibit an increment effect of
NFATe-1, c-Fos and CTSK expressions in the osteoclasts
with time; the results obtained through the laser scanning
confocal microscope show that NaHCO,-TNLs can inhibit
formation of the actin ring of the osteoclasts, indicating that
NaHCO,-TNLs have the significant inhibitory effect on the
bone resorption function of the osteoclasts (as shown in FIG.
3(c)-FIG. 3(e)).

[0087] 4. The receptor activator of nuclear factor-kappa B
(RANK) expressions in the osteoclast inducing system are
evaluated every day. The results obtained through Western-
blot and the laser scanning confocal microscope show that
the RANK expression quantity in the osteoclasts is progres-
sively increased with maturation of the osteoclasts (as
shown in FIG. 3(f) and FIG. 3(g)).

[0088] 5. NaHCO,-TNLs are added into the osteoclast
inducing system and compared with the pure osteoclast
inducing system, and the exosomes are extracted for evalu-
ation. The results obtained through Western-blot and exo-
some flow cytometry show that the RANK content in the
extracellular vesicles is greatly increased with NaHCO,-
TNLs. Compared with the above results, it can be known
that NaHCO,-TNLs can facilitate the osteoclasts to secrete
the RANK-containing extracellular vesicles (as shown in
FIG. 3() and FIG. 3(i)).

[0089] 6. The extracellular vesicles extracted above are
respectively added into the osteoclast inducing systems. The
results obtained through TRAP-staining show that the osteo-
clasts are greatly inhibited with NaHCO,-TNLs induced
extracellular vesicles, indicating that the RANK-containing
extracellular vesicles can further inhibit the osteoclasts (as
shown in FIG. 3(/) and FIG. 3(&)).

[0090] Therapeutic Effects of NaHCO,-TNLs in Example
1 on Osteoporosis of Ovariectomy (OVX) Mice

[0091] 1. Establishing Animal Disease Model and Group-
ing

[0092] Grouping: The 11-week-old C57BL/6 female mice
are divided into four groups, wherein: for mice of the first
group (Sham), a sham operation is conducted, and normal
saline is injected into the caudal veins; for mice of the
second group (OVX), the ovary is removed, and normal
saline is injected into the caudal veins; for mice of the third
group (OVX+NaCl-TNLs), the ovary is removed, and NaCl-
TNLs are injected into the caudal veins; for mice of the
fourth group (OVX+NaHCO,-TNLs), the ovary is removed,
and NaHCO,-TNLs are injected into the caudal veins.
[0093] Implementation: Corresponding operations are
conducted on each group of mice; one week later, corre-
sponding drugs with a dosage of 0.025 ml/g are injected into
the caudal veins every two days, lasting for 2 weeks; 4
weeks after administration is finished, the vertebral body,
femur, tibia and blood of each group of mice are taken out
for analysis. The analysis methods comprise micro-CT,
hematoxylin-eosin (H&E) staining, TRAP staining, and
enzyme-linked immunosorbent assay (ELISA) of serum
bone metabolic indicators (as shown in FIG. 4(a)).

[0094] 2. The micro-CT results of the vertebral body,
femur and tibia of each experimental group are compared.
The results show that the factors of bone volume per tissue
volume (BV/TV), trabecular number (Tb. N) and trabecular

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separation (Tb. Sp) of the OVX+NaHCO,-TNLs group are
all significantly better than that of the OVX group (as shown
in FIG. 4(b) and FIG. 4(c)).

[0095] 3. The H&E staining and TRAP staining results of
the vertebral body, femur and tibia of each experimental
group are compared. The results show that factors of BV/TV,
surface area of the osteoclasts on the bone surface (OC.
S/BS), and number of the osteoclasts on the bone surface
(OC. N/BS) of the OVX+NaHCO,-TNLs group are all
significantly better than that of the OVX group (as shown in
FIG. 4(@) and FIG. 4(e)).

[0096] 4. The serum bone metabolic indicators of each
experimental group are compared. The results show that the
osteoclasts metabolic indicators of the OVX+NaHCO,-
TNLs group are significantly lower than that of the OVX
group. Combined with the above experimental results, it is
indicated that NaHCO,-TNLs can effectively treat bone loss
and osteoclast metabolism of the OVX mice, so as to treat
osteoporosis (as shown in FIG. 4(/)).

[0097] For the nano composite materials aiming at the
acidic sealing zone in the osteoclasts obtained through the
examples 2-6, composite evaluation, characteristic evalua-
tion, evaluation of the inhibitory effects on the osteoclasts,
and evaluation of the therapeutic effects on osteoporosis of
the OVX mice are respectively conducted. The obtained
results are similar to the results of NaHCO,-TNLs in the
example 1, indicating that: through the adjustment of
reagent concentration and treatment time determined by
above optimizations, the preparation of the nano composite
material aiming at the acidic sealing zone in the osteoclasts,
having the similar effect, can be realized.

[0098] It can be known from the above examples that:
through targeting the bone tissues, the nano composite
material aiming at the acidic sealing zone in the osteoclasts
provided by the present invention can conduct an aerogenic
pH response with the acidic sealing zone in the osteoclasts,
and destroy the acidic sealing zone in the osteoclasts while
neutralizing acidification, so that maturation of the osteo-
clasts is inhibited, the osteoclasts are facilitated to secrete
the RANK-containing extracellular vesicles, and ineffective
binding is formed with receptor activator of nuclear factor-
kappa B ligand (RANKL) in serum, thereby achieving the
long-term therapeutic effects on the abnormal osteoclast
activation.

[0099] The above-described is only the examples of the
present invention. Although the present invention is
described in detailed with the above examples, one of
ordinary skill in the art should understand that various
improvements and modifications can be made without
departing from the principle of the present invention. These
improvements and modifications should be all encompassed
in the protection scope of the present invention and in the
scope limited by the claims of the present invention.

Oct. 6, 2022

1. A nano composite material aiming at an acidic sealing
zone in osteoclasts, comprising a nano material, bone-
targeting molecules, and a compound able to react with the
acidic sealing zone in the osteoclasts, wherein: after being
modified by the bone-targeting molecules, the nano material
is loaded with the compound able to react with the acidic
sealing zone in the osteoclasts; the nano material is loadable
and modifiable; the bone-targeting molecules have an obvi-
ous affinity to bone tissues; and the compound able to react
with the acidic sealing zone in the osteoclasts is alkalescent
or neutral bicarbonate.

2. The nano composition material, as recited in claim 1,
wherein: the nano material is liposomes, polymer nano
particles or mesoporous silicon oxide particles.

3. The nano composite material, as recited in claim 1,
wherein: the bone-targeting molecules are tetracycline,
phosphonate or aspartic acid polypeptide sequences.

4. The nano composite material, as recited in claim 1,
wherein: the compound able to react with the acidic sealing
zone in the osteoclasts is sodium bicarbonate, potassium
bicarbonate or ammonium bicarbonate.

5. The nano composite material, as recited in claim 1,
wherein: the compound able to react with the acidic sealing
zone in the osteoclasts is sodium bicarbonate having a
concentration of 1 mol/L.

6. A method for preparing the nano composite material
aiming at the acidic sealing zone in the osteoclasts as recited
in claim 1, comprising steps of: cross-linking the loadable
and modifiable nano material with the bone-targeting mol-
ecules; dissolving in chloroform with lecithin and choles-
terol, and controlling a pH value to 8.0-8.4; at a room
temperature, magnetically stirring and cross-linking for
24-72 hours; forming a membrane in a rotary evaporator;
adding a solution to be loaded, and shaking for hydration;
ultrasonically emulsifying, and dialyzing; wherein: a molar
ratio of the loadable and modifiable nano material to the
bone-targeting molecules is 1:1-1:2.

7. The method, as recited in claim 6, wherein: a func-
tionalized molecule in the loadable and modifiable nano
material is functionalized phospholipid; after cross-linking
with the bone-targeting molecules, bone-targeting function-
alized phospholipid is obtained.

8. The method, as recited in claim 6, wherein: the step of
“ultrasonically emulsifying” specifically comprises steps of:
turning on for 1-2 seconds with a power of 30-70%, then
turning off for 2-3 seconds, and repeating for 5-20 minutes;
and the step of “dialyzing” lasts for 1-3 days.

9. The method, as recited in claim 7, wherein: the func-
tionalized phospholipid is DSPE-PEG-NHS,; and the bone-
targeting molecules are tetracycline (TC).

Pe