U.S. patent application number 11/985263 was filed with the patent office on 2008-06-12 for drug delivery system and method.
Invention is credited to Mark E. Byrne, Siddharth Venkatesh, Jacek Wower.
Application Number | 20080138408 11/985263 |
Document ID | / |
Family ID | 39402250 |
Filed Date | 2008-06-12 |
United States Patent
Application |
20080138408 |
Kind Code |
A1 |
Venkatesh; Siddharth ; et
al. |
June 12, 2008 |
Drug delivery system and method
Abstract
A system for delivering a therapeutic dose of a drug is
disclosed. The system includes a delivery medium with therapeutic
units attached thereto. The delivery medium is preferably a
polymeric hydrogel matrix that has therapeutic units incorporated
therein or metal nanoparticles with therapeutic units complexed
thereto. The therapeutic units include nucleic acid moieties. The
nucleic acid moieties preferably include strands of nucleic acid
and drug moieties complexed with the strands of nucleic acid. Where
the system includes a polymeric hydrogel matrix, an active drug is
controllably released from the polymer hydrogel matrix to provide a
therapeutic dose to a biological system or biological tissue. The
active drug is controllably released from the hydrogel matrix by
altering the environment the hydrogel matrix, or by enzymatic
cleavage of the nucleic acid moieties or by a combination
thereof.
Inventors: |
Venkatesh; Siddharth;
(Auburn, AL) ; Wower; Jacek; (Auburn, AL) ;
Byrne; Mark E.; (Auburn, AL) |
Correspondence
Address: |
HAVERSTOCK & OWENS LLP
162 N WOLFE ROAD
SUNNYVALE
CA
94086
US
|
Family ID: |
39402250 |
Appl. No.: |
11/985263 |
Filed: |
November 13, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60858553 |
Nov 13, 2006 |
|
|
|
60937773 |
Jun 28, 2007 |
|
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|
Current U.S.
Class: |
424/464 ;
424/484; 514/44R |
Current CPC
Class: |
A61K 47/6903 20170801;
A61K 31/70 20130101; C12N 15/87 20130101; A61K 47/6943 20170801;
A61K 48/0091 20130101; A61K 47/6955 20170801; A61K 47/549 20170801;
A61K 48/0008 20130101; A61K 47/58 20170801; A61K 47/34 20130101;
A61K 47/61 20170801 |
Class at
Publication: |
424/464 ;
424/484; 514/44 |
International
Class: |
A61K 9/20 20060101
A61K009/20; A61K 9/00 20060101 A61K009/00; A61K 31/70 20060101
A61K031/70 |
Claims
1. A method for making a drug delivery system, the method
comprising: a) providing backbone monomers, cross-linking monomers
and nucleic acid moieties; and b) initiating copolymerization of
the backbone monomer and cross-linking monomer to thereby form a
polymeric hydrogel with therapeutic units incorporated therein,
wherein the therapeutic units include strands of the nucleic acid
moieties.
2. The method of claim 1, further comprising forming a therapeutic
dose from the polymeric hydrogel.
3. The method of claim 1, wherein forming a polymeric hydrogel
comprises forming a solution comprising an amount of the strands of
nucleic acid, the backbone monomer cross-linking monomer and
initiating copolymerization of the backbone monomer and
cross-linking monomer.
4. The method of claim 1, wherein the nucleic acid moieties include
a drug.
5. The method of claim 4, wherein the drug is selected from the
group consisting of an antibiotic, an anti-inflammatory, an
antihistamine, an antiviral agent, a cancer drug, an anesthetic, a
cycloplegic, a mydriatics, a lubricant agent, a hydrophilic agent,
a decongestant, a vasoconstrictor, a vasodilater, an
Immuno-suppressant, an immuno-modulating agent and an anti-glaucoma
agent.
6. The method of claim 2, wherein the therapeutic dose is in a form
of a pill or a patch.
7. A method of dispensing a therapeutic dose comprising: a)
administering a polymeric hydrogel with therapeutic units
incorporated therein, wherein the therapeutic units include strands
of nucleic acid; and b) initiating a controlled release of portions
of the therapeutic units.
8. The method of claim 7, wherein initiating the controlled release
of the portions of the therapeutic units comprises one or more of
heating the polymeric hydrogel, altering a pH polymeric hydrogel,
altering an ionic strength of the polymeric hydrogel, exposing the
polymeric hydrogel to an electric field and exposing the hydrogel
to light.
9. The method of claim 7, wherein initiating the controlled release
of the portions of the therapeutic units comprises treating the
polymeric hydrogel with an enzyme.
10. The method of claim 7, wherein the polymeric hydrogel is placed
in contact with a target tissue.
11. The method of claim 10, wherein the polymeric hydrogel is
placed in contact with the target tissue by injecting the polymeric
hydrogel into the target tissue.
12. The method of claim 7, wherein initiating the controlled
release of the portions of the therapeutic units comprises treating
the polymeric hydrogel with an enzyme.
13. The method of claim 7, wherein the therapeutic units comprise
one or more active drugs.
14. The method of claim 13, wherein the one or more active drugs
are selected from the group consisting of an antibiotic, an
anti-inflammatory, an antihistamine, an antiviral agent, a cancer
drug, an anesthetic, a cycloplegic, a mydriatics, a vasodilater, a
lubricant agent, a hydrophilic agent, a decongestant, a
vasoconstrictor, an immuno-suppressant, an immuno-modulating agent
and an anti-glaucoma agent.
15. A system for delivering a therapeutic dose of an active drug,
the system comprising a polymeric hydrogel with therapeutic units
incorporated therein, wherein the therapeutic units include strands
of the nucleic acid.
16. The system of claim 15, wherein therapeutic units include a
drug moiety complex with the strands of nucleic acid.
17. The system of claim 16, wherein the drug is selected from the
group consisting of an antibiotic, an anti-inflammatory, an
antihistamine, an antiviral agent, a cancer drug, an anesthetic, a
cycloplegic, a mydriatics, a lubricant agent, a hydrophilic agent,
a decongestant, a vasoconstrictor, a vasodilater, an
Immuno-suppressant, an immuno-modulating agent and an anti-glaucoma
agent.
18. The system of claim 15, wherein the polymeric hydrogel is in a
pill or a patch form.
19. The system of claim 15, wherein the therapeutic dose of a drug
is controllably released from the polymeric hydrogel by enzymatic
cleavage of the strands of the nucleic acid moieties.
20. A system for delivering a therapeutic dose of an active drug,
the system comprising a delivery medium with therapeutic units
incorporated therein, wherein the therapeutic units include strands
of the nucleic acid.
21. The system of claim 20, wherein the delivery medium includes
one or more of a gel matrix, metal particles, a polymer film, a
polymer network, metal surface, polymer particles, particulate
gels, particulate networks, a polymeric dendrimer and a surface
conjugated with complexes.
22. The system of claim 20, wherein the delivery medium comprises
metal nanoparticles with therapeutic nucleic acid strands and
receptor moieties coupled thereto.
23. The system of claim 22, wherein the metal nanoparticles are
silver or gold nanoparticles.
Description
RELATED APPLICATIONS
[0001] This application claims priority under 35 U.S.C. .sctn.
119(e) of the co-pending U.S. Provisional Application Ser. No.
60/858,553, titled "Hypersensitive Modulating Elements for
Therapeutic Delivery," filed Nov. 13, 2006 and the co-pending U.S.
Provisional Application Ser. No. 60/937,773, titled "Drug Delivery
Vehicles for On-Demand Therapeutic Release," filed Jun. 28, 2007.
The co-pending U.S. Provisional Application Ser. No. 60/858,553,
titled "Hypersensitive Modulating Elements for Therapeutic
Delivery," filed Nov. 13, 2006 and the co-pending U.S. Provisional
Application Ser. No. 60/937,773, titled "Drug Delivery Vehicles for
On-Demand Therapeutic Release," filed Jun. 28, 2007 are both hereby
incorporated by reference.
FIELD OF THE INVENTION
[0002] The invention relates to drug delivery systems. More
specifically, this invention relates to systems for and methods of
controlled drug delivery using a delivery medium and nucleic acids
incorporated therein.
BACKGROUND OF THE INVENTION
[0003] Routes of administration of drugs are commonly administered
via topical, enteral (via the digestive tract), parenteral routes
(injection or infusion). Topical generally refers to, but is not
limited to epicutaneous, inhalational, intranasal, vaginal, ocular
surface and ear drops. Enteral generally refers to, but is not
limited to the digestive tract, oral cavity, gastric cavity, and
rectal administration. Parenteral generally refers to, but is not
limited to administration by injection or infusion via intravenous,
intrarterial, intramuscular, subcutaneous, transdermal,
transmucosal, intradermal, intrathecal, intraosseous, intracardiac,
interperitoneal, intravitreal, and inhalational.
[0004] RNA is emerging as an important drug target and versatile
therapeutic agent because it folds into complex 3-D structures
capable of expressing many enzymatic activities and because it
"digitally" interferes with the flow of genetic information from
DNA to proteins. Recent studies demonstrated that RNA also
constitutes an attractive material for nanotechnology because RNA
molecules can be easily programmed to carry out specific functions
through the incorporation of aptamers. These novel "smart"
macromolecules can be selected from random pools of RNA molecules
based on their ability to bind metals, small organic compounds,
nucleic acids, proteins and even entire cells and to change their
inherent resistance to degradation for our benefit.
[0005] Aptamers can be produced by a conceptually straightforward
two-step process that involves in vitro synthesis of more than
10.sup.15 individual RNA molecules and screening them by column
affinity chromatography. This approach is commonly known as in
vitro selection or Systemic Evolution of Ligands by EXpotential
enrichment (SELEX). Although RNA is sensitive to degradation by
ribonucleases (RNases), its stability can be easily regulated by
incorporation of modified nucleotides. For example, incorporation
of fluorine-CTP and -UTP (Epicentre Bio-technologies) makes RNA
resistant to degradation by ubiquitous RNase A.
[0006] Another group of "smart" RNA molecules, ribozymes, are able
to catalyze fundamental biological processes such as the synthesis
of proteins (transpeptidation), aminoacylation of tRNA molecules
(esterification), and RNA cleavage (transesterification). Their
discovery has changed our views of macromolecular evolution,
recognizing the fact that an informational molecule can
simultaneously posses enzymatic activity. Ribozymes have now been
described in a number of systems from bacteria through humans. The
ubiquity of catalytic RNAs has prompted intensive investigation
into potential applications as well as the mechanism of catalysis.
The catalytic performance of nucleic acids can be enhanced by the
incorporation of additional functional groups. A number of new
ribozymes was discovered using SELEX.
SUMMARY OF THE INVENTION
[0007] The present invention is directed to methods of and system
for delivering a therapeutic dose of a drug. A system in accordance
with the present invention includes a delivery medium with
therapeutic units attached thereto. The delivery medium is any
suitable delivery medium that can complex or incorporate the
therapeutic units. For example, the delivery medium is a gel
matrix, metal particles, a polymer film, a polymer network, a
metal, a polymer particle, particulate gels, particulate networks,
a polymeric dendrimer, a surface conjugated with complexes or any
combination thereof. The therapeutic units include nucleic acid
moieties with active drug portions that are controllably released
to provide a therapeutic dose to a biological system or biological
tissue.
[0008] Nucleic acid (NA) is a single or double stranded polymer or
oligomer consisting of ribonucleotides, deoxyribonucleotides or
their modified derivatives such as fluorinated RNA, methylated DNA,
locked nucleic acids (LNA), peptide nucleic acids (PNA). The
Nucleic acids used in the present invention can be selected or
designed. NA strands can interact to form double strands or to
direct NA strands towards the molecular target using hybridization
(anti-sense mechanism).
[0009] The term "active drug" herein refers broadly to a molecular
species that provides a therapeutic result when administered to a
biological system or tissue. An active drug is, for example, an
antibiotic, an anti-inflammatory, an antihistamine, an antiviral
agent, a cancer drug, an anesthetic, a cycloplegic, a mydriatics, a
lubricant agent, a hydrophilic agent, a decongestant, a
vasoconstrictor, a vasodilater, an Immuno-suppressant, an
immuno-modulating agent, an anti-glaucoma agent or a combination
thereof. In accordance with the embodiments of the present
invention, an active drug is also in a form of strands or fragments
of RNA (ribonucleic acid) and/or DNA (deoxyribonucleic acid). Drug
herein also refers to prodrugs, nucleic acids, glyocproteins, lipid
conjugated drugs and protein-based drugs.
[0010] A drug herein also includes Pharmacologically active agents
and can include anti-cancer drugs, analgesics, antipyretics,
nonsteriodal anti-inflammatory drugs, steroidal anti-inflammatory
drugs, anti-allergics, anti-histamines, anti-bacterial drugs,
antibiotics, anti-infective drugs, anti-fungal drugs, statins,
anti-anemia drugs, cytotoxic drugs, anti-hypertensive drugs,
cholesterol lowering medications, dermatological drugs,
psychotherapeutic drugs, vitamins, minerals, dietetics,
anti-adiposity drugs, carbohydrate metabolism drugs, protein
metabolism drugs, thyroid drugs, antithyroid drugs, anti-macular
degeneration drugs, anti-retinal degenerative disease drugs,
anti-diabetic retinopathydrugs, anti-uveitis drugs, anti-glaucoma
drugs, immuno-modulating agents, anti-viral agents, coenzymes and
combinations thereof.
[0011] In accordance with an embodiment of the invention, a system
for delivering a therapeutic dose of an active drug includes a
polymeric hydrogel matrix. The polymeric hydrogel matrix has
therapeutic units incorporated therein. The therapeutic units
include strands of the NA. The strands of the NA or portions
thereof can act as the active drug when they are released from the
polymeric hydrogel matrix. In further embodiments the therapeutic
units include strands of NA moieties. As used herein, "NA moieties"
refers to strands of the NA that have drug moieties selectively
bound to the stands of NA. In this embodiment the active drug
includes the drug moiety or any portion of the NA moiety that is
released from the polymeric hydrogel matrix to provide a
therapeutic dose. In use, the NA moieties are preferably
controllably and/or selectively cleaved and/or released from
polymeric hydrogel matrix to thereby release the active drug
portions. It will be clear to one skilled in the art from the
discussion below that a drug delivery system of the present
invention with different types of therapeutic units with, for
example, more than one type of NA moiety and/or more than one type
of drug moiety is within the scope of the preset invention. A drug
delivery system with different types of therapeutic units is useful
to provide a controlled release of multiple active drugs in
different quantities and/or at different rates.
[0012] In accordance with the embodiments of the invention, a
system for delivering a therapeutic dose of a drug includes metal
nanoparticles. The metal nanoparticles are metal particles that
have an average particle size in a range of 5 to 100 nanometers in
diameter. The metal nanoparticles are formed from any suitable
metal or combination of metals, but are preferably formed from a
metal that is nontoxic or exhibits minimal toxicity to a biological
system or tissue being treated. For example, the metal
nanoparticles are formed from silver or gold. The metal
nanoparticles are functionalized with therapeutic NA moieties, such
as described above, and other moieties that solubilize the metal
nanoparticles and/or receptor moieties that target biological
tissues or molecules.
[0013] The drug delivery system of the present can take any number
of forms. For example, the system can be in a pill form, a patch
form or a liquid form. As used herein "patch form" includes a
contact lense or any other form of a drug delivery system that
administers a drug through extended contact with a biological
tissue. Further details of therapeutic contact lenses are provided
in the U.S. patent application Ser. No. 11/346,770, filed Feb. 3,
2006, and titled "Contact Drug Delivery System," the contents of
which are hereby incorporated by reference.
[0014] In use, a drug delivery system in accordance with the
present invention is taken orally, intravenously or absorbed
through contact with a biological tissue. For example, the drug
delivery system can be injected at or near a target tissue to be
treated. Once the drug delivery system is taken, the polymeric
hydrogel matrix with the therapeutic units provides a controlled
release of an active drug. The controlled release of the active
drug is achieved by altering an environment of the polymeric
hydrogel matrix. For example, the polymeric hydrogel matrix is
heated, treated with a pH modifier, treated with an ionic solution,
exposed to an electric field and/or exposed to light. In this way
the release of the active drug can be stopped or started.
Alternatively, the release of the active drug is controlled over
time at a rate that is regulated by a degradation rate of the NA
moieties and the folding of the NA moieties in the therapeutic
units. In a particular embodiment of the present invention, a
controlled release of an active drug is achieved by treating the
polymeric hydrogel matrix with an enzyme that selectively cleaves
the strands of the NA moieties and, thereby, releases active drug
portions of the therapeutic units.
[0015] In accordance with the embodiments of the present invention,
a drug delivery system with polymeric hydrogel matrix and
therapeutic units incorporated therein is formed by providing a
backbone monomer, a cross-linking monomer and NA moieties, and
initiating copolymerization of the backbone monomer and
cross-linking monomer. The NA moieties are NA strands and/or NA
strands with drug moieties complexed thereto. In accordance with an
embodiment of the present invention NA strands are complexed with
drug moieties prior to initiating copolymerization of the backbone
monomer and cross-linking monomer, in situ with the formation of
polymeric hydrogel matrix or after the polymeric hydrogel matrix is
formed. The backbone functional groups in the polymeric hydrogel
matrix are not required to interact with the drug, but rather
provide a matrix to host the drug and control its release.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1A is a schematic representation showing the formation
of a drug delivery system with a polymeric hydrogel matrix with
therapeutic units that include NA moieties, portions of which are
released by enzymatic cleavage, in accordance with the embodiments
of the invention.
[0017] FIG. 1B illustrates a graphical representation of a
controlled release profile of active drug portions of NA fragments
from a drug delivery system by enzymatic cleavage within a hydrogel
matrix, in accordance with the embodiments of the invention.
[0018] FIG. 2A is a schematic representation showing the formation
of a drug delivery system with a polymeric hydrogel with
therapeutic units that include strands of the NA and drug moieties
complexed with strands of NA and that are released by enzymatic
cleavage, in accordance with the embodiments of the invention.
[0019] FIG. 2B illustrates a graphical representation of a
controlled release profile of drug moieties from strands of NA in a
hydrogel matrix, in accordance with the embodiments of the
invention.
[0020] FIG. 3 is a schematic representation showing the formation
of a drug delivery system with a polymeric hydrogel and therapeutic
units that include strands of the NA that are released by altering
the environment of the polymeric hydrogel, in accordance with the
embodiments of the invention.
[0021] FIG. 4A is a schematic representation showing the formation
of a drug delivery system with metal nanoparticles that are
functionalized with receptor molecules and two different
therapeutic units each including NA moieties, in accordance with
the embodiments of the invention.
[0022] FIG. 4B illustrates a graphical representation of controlled
release profiles for two different active drugs released from the
drug delivery system represented in FIG. 4A, in accordance with the
embodiments of the invention.
[0023] FIG. 5 shows Structure and Function of an Intelligent
Biohybrid Hydrogel with RNA Crosslinks.
[0024] FIGS. 6A-B show Dynamic Tunable Release Profiles of DNA from
Novel Biohybrid Gels by Enzymatic or Temperature Triggers.
[0025] FIGS. 7A-B show Tunable Release Profiles of DNA from
Biocompatible Gels by Controlling Macromolecular Architecture and
Enzymatic Triggers.
DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT
[0026] FIG. 1A is a schematic representation 100 showing the
formation of a drug delivery system 111 with a polymeric hydrogel
matrix with therapeutic units 121 incorporated therein. The drug
delivery system 111 is formed by providing backbone monomers 101,
103, 105, a cross-linking monomer (not shown), and therapeutic
units 121, and initiating copolymerization of the backbone monomers
101, 103, 105 and the cross-linking monomer. The therapeutic units
121 are incorporated into the polymeric hydrogel matrix during the
formation of the polymeric hydrogel matrix or can be incorporated
into the polymeric hydrogel matrix after the polymeric hydrogel
matrix is formed.
[0027] Polymeric hydrogel matrix of the present are incorporated
into electrophoresis wells or used as membranes, films, etc. to add
in the separation and capture of nucleic acid sequences.
Electrophoresis and diffusion can both be used to construct a
nucleic acid:drug complex within the gel or to remove
unincorporated nucleic acid and drug moieties.
[0028] In accordance with the embodiments of the invention, a
polymeric hydrogel matrix is formed from silicon-based
cross-linking monomers, carbon-based or organic-based monomers,
macromers or combinations thereof. Suitable cross-linking monomers
include but are not limited to Polyethylene glycol (200)
dimethacrylate (PEG200DMA), ethylene glycol dimethacrylate (EGDMA),
tetraethyleneglycol dimethacrylate (TEGDMA),
N,N'-Methylene-bis-acrylamide and polyethylene glycol (600)
dimethacrylate (PEG600DMA). Suitable silicon-based cross-linking
monomers include, but are not limited to,
tris(trimethylsiloxy)silyl propyl methacrylate (TRIS) and
hydrophilic TRIS derivatives such as tris(trimethylsiloxy)silyl
propyl vinyl carbamate (TPVC), tris(trimethylsiloxy)silyl propyl
glycerol methacrylate (SIGMA), tris(trimethylsiloxy)silyl propyl
methacryloxyethylcarbamate (TSMC); polydimethylsiloxane (PDMS) and
PDMS derivatives, such as methacrylate end-capped fluoro-grafted
PDMS crosslinker, a methacrylate end-capped urethane-siloxane
copolymer crosslinker, a styrene-capped siloxane polymer containing
polyethylene oxide and polypropylene oxide blocks; and siloxanes
containing hydrophilic grafts or amino acid residue grafts, and
siloxanes containing hydrophilic blocks or containing amino acid
residue grafts. The molecular structure of these monomers can be
altered chemically to contain moieties that match amino acid
residues or other biological molecules. In cases where the above
monomers are polymerized with hydrophilic monomers, a solubilizing
cosolvent may be used such as dimethylsulfoxide (DMSO),
isopropanol, water, alchohol, or water/alchohol mixtures.
[0029] Preferably, crosslinkers contain more than one vinyl group
in the structure or chemistry to covalently bond with multiple
monomeric or oligomeric structures. Degradable crosslinkers and
grafted structures degrade at a characteristic rate and include,
but are not limited to poly lactic acid and polyglycolic acid
macromers and derivatives, degradable thiol-ene polymers and
etc.
[0030] Crosslinking monomer amounts from 0.01 to 90%, backbone
monomers from 99.99% to 10% (moles backbone monomers/moles total
monomers) with varying relative portions of backbone monomers (some
of which may be functional and interact with the drug, nucleic
acid, or both); initiator concentrations ranging from 0.1 to 30 wt
%; solvent concentrations ranging from 0% to 80 wt %; monomers to
therapeutic unit ratios ranging from 0.001 to 5,000, optimized to
give a high therapeutic unit loading (therapeutic unit may be
linked to network via covalent or non-covalent chemistry or contain
an acrylate or methacrylate group and link to the network as other
monomers in the polymerization reaction).
[0031] Still referring to FIG. 1, in accordance with a method of
the present invention, the drug delivery system 111 is formed by
making a mixture or solution that includes amounts of the
therapeutic units 121, the backbone monomers 101, 103 and 105, the
cross-linking monomer and a polymerization initiator in a suitable
solvent or without a solvent. Suitable initiators include water and
non-water soluble initiators include, but are not limited to, TEMED
(N,N,N,N-Tetramethyl-Ethylenediamine) or other reaction accelerator
in conjunction with ammonium persulfate, azobisisobutyronitrile
(AIBN), 2,2-dimethoxy-2-phenyl acetophenone (DMPA),
1-hydroxycyclohexyl phenyl ketone (Irgacureo 184),
2,2-dimethoxy-1,2-diphenylethan-1-one (Irgacure 651), ammonium
persulfate, iniferters such as tetraethylthiuram disulfide, or
combinations thereof for living or controlled polymerization
methods. The polymerization is able to be photo-initiated,
thermally-initiated, redox-initiated or combinations thereof.
[0032] Examples of living or controlled polymerization include, but
are not limited to living anionic or cationic polymerization, ring
opening metathesis polymerization (ROMP), group transfer
polymerization (GOP), living Ziegler-Natta polymerization, and
free-radical polymerization (e.g., iniferter polymerization,
catalytic chain transfer polymerization, stable free radical
mediated polymerization (SFRP), ATRF or atom transfer radical
polymerization, reversible addition fragmentation chain transfer
(RAFT) polymerization, Iodine Transfer polymerization,
Selenium-centered mediated polymerization, Telluride-mediated
polymerization (TERP), Stibine-mediated polymerization).
[0033] The backbone monomers 101, 103 and 105 complex with the
therapeutic units 121 and copolymerize with the cross-linking
monomer to form the drug delivery system 111, such as described
above. Alternatively, the monomers 101, 103 and 105 do not complex
or weakly complex with the therapeutic units 121 and copolymerize
with the cross-linking monomer to form the drug delivery system
111. Functional or reactive monomers 101 and 103 useful herein are
those which possess chemical or thermodynamic compatibilities with
a desired therapeutic unit 121. As used herein, the term backbone
monomer includes moieties or chemical compounds that have at least
one double bond group that can be incorporated into a growing
polymer chain by chemical reaction and that have one end that will
functionally interact with the therapeutic unit 121 through one or
more of electrostatic interactions, hydrogen bonding, hydrophobic
interactions, coordination complexation, and Van der Waals forces.
Backbone monomers include macromers, oligomers, and polymer chains
with pendent functionality and which have the capability of being
crosslinked to create the recognitive hydrogel. Crosslinking
monomers include chemicals with multiple double bond functionality
that can be polymerized into a polymer network.
[0034] Examples of backbone monomers include, but are not limited
to, 2-hydroxyethylmethacrylate (HEMA), Acrylic Acid (AA),
Acrylamide (AM), N-vinyl 2-pyrrolidone (NVP), 1-vinyl-2-pyrrolidone
(VP), methyl methacrylate (MMA), methacrylic acid (MAA), acetone
acrylamide, 2-ethyl-2-(hydroxymethyl)-1,3-propanediol
trimethacrylate, N-(1,1-dimethyl-3-oxobutyl)acrylamide,
2-ethyl-2-(hydroxymethyl)-1,3-propanediol trimethacrylate,
2,3-dihydroxypropyl methacrylate, allyl methacrylate,
3-[3,3,5,5,5-pentamethyl-1,1-bis[pentamethyldisiloxanyl)oxy]trisiloxanyl]-
propyl methacrylate,
3-[3,3,3-trimethyl-1,1-bis(trimethylsiloxy)disiloxanyl]propyl
methacrylate (TRIS), N-(1,1-dimethyl-3-oxybutyl)acrylamide,
dimethyl itaconate, 2,2,2,-trifluoro-1-(trifluoromethyl)ethyl
methacrylate, 2,2,2-trifluoroethyl methacrylate,
methacryloxypropylbis(trimethylsiloxy)methylsilane,
methacryloxypropylpentamethyldisiloxane,
(3-methacryloxy-2-hydroxypropyloxy)propylbis(trimethylsiloxy)methylsilane-
, 4-t-butyl-2-hydroxycyclohexyl methacrylate, dimethylacrylamide,
glycerol methacrylate and diethylaminoethyl methacrylate
(DEAEM).
[0035] Once the drug delivery system 111 is formed, it is fashioned
into a pill, a contact lens, a patch or any other suitable form
that allows it to be delivered to a biological system or biological
tissue. The system 111 is formed outside or inside the body using
various methods to produce films, macroflims, microfilms,
nanofilms, irregular particles and other shapes, surface coatings,
particles, contact lenses with curved surfaces, etc.
[0036] Still referring to FIG. 1A, the therapeutic units 121
preferably include NA moieties 123, 123' and 123''. The NA moieties
include strands of NA with active drug portions 113. In use the
drug delivery system 111 is delivered to a biological system or
biological tissue using any suitable method. The drug delivery
system 111 is then treated with an enzyme. The enzyme selectively
cleaves the NA moieties 123, 123' and 123'' and releases the active
drug portions 113, thereby administering a therapeutic dose of the
active drug portions 113 to the biological system or biological
tissue.
[0037] FIG. 1B illustrates a graphical representation 150 of a
controlled release profile 151 of the active drug portions 113 from
the drug delivery system 111 by enzymatic cleavage, in accordance
with the embodiments of the invention. After the therapeutic dose
of the active drug portion 113 is released to the biological system
or biological tissue, the depleted hydrogel matrix 111' (FIG. 1A)
preferably degrades within the biological system or biological
tissue. The system is also controllable, such that a release
profile 151'' of the active drug portions 113 from the drug
delivery system 111 is constant.
[0038] FIG. 2A is a schematic representation 200 showing the
formation of a drug delivery system 211, in accordance with further
embodiments of the invention. The drug delivery system 211 with a
polymeric hydrogel matrix further includes therapeutic units 222
incorporated into the polymeric hydrogel matrix. The drug delivery
system 211 is formed by providing backbone monomers 201, 203 and
205, a cross-linking monomer (not shown) and therapeutic units 222,
and initiating copolymerization of the backbone monomers 201, 203
and 205 and cross-linking monomer with a suitable initiator, such
as described above.
[0039] Still referring to FIG. 2A, the therapeutic units 222
preferably include NA moieties 223. The NA moieties 223 include
strands of NA such as described previously. The therapeutic units
222 further include drug moieties 213 that are complexed with the
NA moieties 223. The drug moieties 213 are suitable drug molecules
or a combination of drug molecules. In accordance with the
embodiments of the invention, multiple drug moieties are used to
administer therapeutic doses of the multiple drug moieties from a
single drug delivery system 211.
[0040] Suitable drug moieties include, but are not limited to,
Anti-bacterials, Anti-infectives, Anti-microbial Agents, such as
anti-fungal agents (all of which generally referred to as
antibiotics) such as Penicillins (including Aminopenicillins and/or
penicillinas in conjunction with penicillinase inhibitor and
anti-fugal agents), Cephalosporins (and the closely related
cephamycins and carbapenems), Fluoroquinolones, Tetracyclines,
Macrolides, Aminoglycosides. Specific examples include, but are not
limited to, erythromycin, bacitracin zinc, polymyxin, polymyxin B
sulfates, neomycin, gentamycin, tobramycin, gramicidin,
ciprofloxacin, trimethoprim, ofloxacin, levofloxacin, gatifloxacin,
moxifloxacin, norfloxacin, sodium sulfacetamide, chloramphenicol,
tetracycline, azithromycin, clarithyromycin, trimethoprim sulfate
and bacitracin.
[0041] Drug delivery systems of the present invention are used to
deliver Non-steroidal (NSAIDs) and Steroidal Anti-inflammatory
Agents (generally referred to as anti-inflammatory agents)
including both COX-1 and COX-2 inhibitors. Examples include, but
are not limited to, corticosteroids, medrysone, prednisolone,
prednisolone acetate, prednisolone sodium phosphate,
fluormetholone, dexamethasone, dexamethasone sodium phosphate,
betamethasone, fluoromethasone, antazoline, fluorometholone
acetate, rimexolone, loteprednol etabonate, diclofenac (diclofenac
sodium), ketorolac, ketorolac tromethamine, hydrocortisone,
bromfenac, flurbiprofen, antazoline and xylometazoline.
[0042] Drug delivery systems of the present invention are also used
to deliver Anti-histamines, Mast cell stabilizers, and Anti-allergy
Agents (generally referred to as anti-histamines). Examples
include, but are not limited, cromolyn sodium, lodoxamide
tromethamine, olopatadine HCl, nedocromil sodium, ketotifen
fumurate, levocabastine HCL, azelastine HCL, pemirolast (pemirolast
potassium), epinastine HCL, naphazoline HCL, emedastine,
antazoline, pheniramine, sodium cromoglycate, N-acetyl-aspartyl
glutamic acid and amlexanox.
[0043] In yet further embodiments of the invention, the drug
delivery systems of the present invention are used to deliver
Anti-viral Agents including, but not limited to, trifluridine and
vidarabine; Anti-Cancer Therapeutics including, but not limited to,
dexamethasone and 5-fluorouracil (5FU); Local Anesthetics
including, but are not limited to, tetracaine, proparacaine HCL and
benoxinate HCL; Cycloplegics and Mydriatics including, but not
limited to, Atropine sulfate, phenylephrine HCL, Cyclopentolate
HCL, scopolamine HBr, homatropine HBr, tropicamide and
hydroxyamphetamine Hbr; Comfort Molecules or Molecules (generally
referred as lubricating agents) to treat Keratoconjunctivitis Sicca
(Dry Eye) including, but not limited to, Hyaluronic acid or
hyaluronan (of varying Molecular Weight, MW), hydroxypropyl
cellulose (of varying MW), gefarnate, hydroxyeicosatetranenoic acid
(15-(S)-HETE), phospholipid-HETE derivatives, phoshoroylcholine or
other polar lipids, carboxymethyl cellulose (of varying MW),
polyethylene glycol (of varying MW), polyvinyl alcohol (of varying
MW), rebamipide, pimecrolimus, ecabet sodium and hydrophilic
polymers; Immuno-suppressive and Immuno-modulating Agents
including, but not limited to, Cyclosporine, tacrolimus, anti-IgE
and cytokine antagonists; and Anti-Glaucoma Agents including beta
blockers, pilocarpine, direct-acting miotics, prostagladins, alpha
adrenergic agonists, carbonic anhydrase inhibitors including, but
not limited to betaxolol HCL, levobunolol HCL, metipranolol HCL,
timolol maleate or hemihydrate, carteolol HCL, carbachol,
pilocarpine HCL, latanoprost, bimatoprost, travoprost, brimonidine
tartrate, apraclonidine HCL, brinzolamide and dorzolamide HCL;
decongestants, vasodilaters vasoconstrictors including, but not
limited to epinephrine and pseudoephedrine.
[0044] Still referring to FIG. 2A, in use, the drug delivery system
211 is delivered to a biological system or biological tissue using
any suitable method. The drug delivery system 211 is then treated
with an enzyme, a denaturing agent or any other suitable chemical
that causes the complexed drug moieties 213 to be controllably
released from the polymeric hydrogel matrix, to thereby
administering a therapeutic dose of the drug moieties 213 to the
biological system or biological tissue.
[0045] FIG. 2B illustrates a graphical representation 250 of a
controlled release profile 251 of the drug moieties 213 from the
drug delivery system 211 (FIG. 2A), in accordance with the
embodiments of the invention. After the therapeutic dose of the
drug moieties 213 is released to the biological system or
biological tissue, the depleted hydrogel matrix 211' preferably
degrades within the biological system or biological tissue. The
system is also controllable, such that a release profile 251'' of
the active drug portions 213 from the drug delivery system 211 is
constant
[0046] It will be clear to one skilled in the art that a controlled
release of the active drug portions 113 (FIG. 1A) and/or drug
moieties 213 (FIG. 2A) can be achieved by altering an environment
of the polymeric hydrogel matrix in any number of different ways.
For example, the polymeric hydrogel matrix can be heated, treated
with a pH modifier, treated with an ionic solution, exposed to
light to controllably release the active drug portions 113 and/or
drug moieties 213. Alternatively, the active drug portions 113
and/or drug moieties 213 are released through bio-degradation of
the NA moieties within a biological system or biological tissue.
Also and described previously, controlled release of the active
drug portions 113 and/or drug moieties 213 can be achieved by
degradation of any portion of the systems.
[0047] The release of the complexed drug moieties 213 (FIG. 2A) can
be controlled through the affinity of the drug moieties 213 with
the NA moieties 223. For example, a strongly complexed drug
moieties 213 will be released from the system 211 at a slower rate
than that of a weakly complexed drug moieties 213. In some cases
where drug moieties 213 are very strongly complexed, the drug
moieties 213 will only be released from the system by degradation
of the NA moieties 223. If, however, drug moieties 213 are very
weakly complexed, the drug moieties 213 will be released from the
system 211 based primarily on the concentration gradient of the
drug moieties 213.
[0048] Also, NA aptmer-drug complexes in accordance with the
embodiment of the invention are noncovalently linked and/or
dispersed within a gel structure. Release of therapeutic can be
controlled by the macromolecular architecture. For instance, a
tight crosslinking structure will lead to an increased transport of
therapeutic from the structure but significantly deter the NA
sequences depending on the size of these structures. The affinity
of the NA for the therapeutic along with the structure of the gel
will determine the release rate.
[0049] FIG. 3 is a schematic representation 300 showing the
formation of a drug delivery system 303 with a polymeric hydrogel
matrix 301 and therapeutic units 309 that include strands of NA.
The therapeutic units 309 are incorporated into the polymeric
hydrogel matrix 301 after the polymeric hydrogel matrix 301 is
formed. The therapeutic units 309 are incorporated into the
polymeric hydrogel matrix 301 by treating the polymeric hydrogel
matrix 301 with the therapeutic units 309 in an ionic environment
to form the drug delivery system 303. The ionic environment can,
for example, be a solution of magnesium ions or any other suitable
solution of metal ions.
[0050] Still referring to FIG. 3, in use, the drug delivery system
303 is delivered to a biological system or biological tissue using
any suitable method. The drug delivery system 303 is then treated
with a chelating agent, such as ethylenediamine tetraacetate, that
chelates the metal ions and, thereby, releases the therapeutic
units 309 from the polymeric hydrogel matrix 301 and into the
biological system or biological tissue being treated.
[0051] FIG. 4A is a schematic representation 400 showing the
formation of a drug delivery system 401 with metal nanoparticles
that are functionalized with receptor molecules 431 and therapeutic
units 421. The receptor molecules or ligand molecules 431 are
provided to target specific cells in the body. The particle surface
can also be passivated using polyethylene glycol to evade immune
surveillance and the reticuloendothelial system (RES) or extend
circulation time (delay excretion).
[0052] The metal nanoparticles are metal particles that have an
average particle size in a range of 5 to 100 nanometers in diameter
and are formed from silver or gold or a combination thereof. Larger
metal nanoparticles greater than 100 nanometers are considered to
within the scope of the invention.
[0053] In accordance with the embodiments of this invention, the
therapeutic units 421 include at least two different therapeutic
units 421' and 421'' each including NA moieties or NA moieties with
drug moieties complexed thereto. The different therapeutic units
421' and 421'' are complexed to the metal nanoparticles through any
number of chemical processes. For example the different therapeutic
units 421' and 421'' are tailored with a thiol group that bond to a
surface of the metal nanoparticles. The receptor molecules 431 are
provided to solubilize the metal nanoparticles and/or target
specific biological tissues.
[0054] Still referring to FIG. 4A, in use, the drug delivery system
401 is delivered to a biological system or biological tissue using
any suitable method. The drug delivery system 401 or the
environment of the drug delivery system 401 is then altered
chemically, photo-chemically or physically, such that the different
therapeutic units 421' and 421'' or portions of the different
therapeutic units 421' and 421'' (active drug portions) are
released from the metal nanoparticles and into the biological
system or biological tissue being treated. Alternatively, the
different therapeutic units 421' and 421'' or portions of the
different therapeutic units 421' and 421'' are released from the
metal nanoparticles through biodegradation of the NA portions of
the different therapeutic units 421' and 421'' within the
biological environment being treated. Release of the different
therapeutic units 421' and 421'' can also occur from unfolding of
the RNA, degradation of the RNA, or if the affinity of the nucleic
acid-drug complex optimized-release by a concentration gradient.
The affinity of the nucleic acid for drug can be controlled and
selected.
[0055] FIG. 4B illustrates a graphical representation 450 of
controlled release profiles 451 and 451' of two different
therapeutic units 421' and 421'' or portions of the two different
therapeutic units 421' and 421'' as they are released from the drug
delivery system 401 represented in FIG. 4A, in accordance with the
embodiments of the invention. From the controlled release profiles
451 and 451' it can be seen that the different therapeutic units
421' and 421'' or portions of the two different therapeutic units
421' and 421'' are released at different rates. In this way a
therapeutic dose of a drug cocktail is able to be administered to a
biological system or biological tissue.
[0056] FIG. 5 represents a Reversible Effective Crosslinking within
gel by an NA aptamer binding molecule or NA moiety 513. A
conjugated or covalently attached molecule (which could be a sugar,
therapeutic, biological marker, antigen, antibody, or other
biologically significant moiety) to polymer chains can complex with
a conjugated NA aptamer and provide reversible effective crosslinks
in the gel. A dispersed therapeutic 523 in the gel can then be
modulated in release. When the complex is formed, transport of drug
523 is reduced and when the complex 511 is not formed the transport
of drug is significantly increased leading to on-off modulated
delivery. Also, if a free molecule enters the gel that is similar
to or the same as the covalently attached molecule, release of the
dispersed therapeutic will be concentration dependent and triggered
by the free molecule. After the drug 523 is released, the depleted
polymer 511' preferably biodegrades.
[0057] A conjugated or covalently attached molecule (which could be
a sugar, therapeutic, biological marker, antigen, antibody, or
other biologically significant moiety) to polymer chains can
complex with a conjugated NA aptamer and provide reversible
effective crosslinks in the gel. A dispersed therapeutic in the gel
can then be modulated in release. When the complex is formed
transport of drug is reduced and when the complex is not formed or
inhibited the transport of drug is significantly increased leading
to on-off modulated delivery. Also, if a free molecule enters the
gel that is similar to or the same as the covalently attached
molecule, release of the dispersed therapeutic will be
concentration dependent and triggered by the free molecule.
[0058] Despite entrapment in the network, the DNA helix can be
readily cleaved by restriction enzyme, leading to controlled
release of the cleaved DNA strands (FIG. 6A). FIG. 6A and FIG. 6B
show Dynamic Tunable Release Profiles of DNA from Novel Biohybrid
Gels by Enzymatic or Temperature triggers. FIG. 6A illustrates a
graph 600 showing the tailored release of DNA by incubating the
gels in BamHI at physiological conditions, as illustrated by the
line 610, as compared to incubating in the buffer only, as
illustrated by the line 620. FIG. 6B illustrates a graph 650
showing the sigmoidal temperature dependent release of DNA, as
illustrated by the line 660. FIG. 6B highlights the temperature
responsive release characteristics which match the theoretical
melting temperature (58.degree. C.). Since melting temperature is a
direct function of G-C pairs, this will provide the opportunity to
tune the release characteristics by varying the sequence.
Optimization of the macromolecular structure of the network or the
DNA molecules will lead to programmable release profiles.
[0059] FIG. 7A and FIG. 7B show Tunable Release Profiles of DNA
from Biocompatible Gels by Controlling Macromolecular Architecture
and Enzymatic Triggers. FIG. 7A illustrates a graph 700 showing the
tailored release of DNA by incubating the gels in DNase I at
physiological conditions in biocompatible gels of varying
crosslinking densities. Crosslinking densities, calculated on an
acrylated DNA-free basis, was 0.15%, as illustrated by the line
710, 0.22%, as illustrated by the line 711, and 0.45%, as
illustrated by the line 712. FIG. 7B illustrates a graph 750
showing the tailored release of DNA by incubating a gel of weak
crosslinking density (0.15%) in DNase I at physiological
conditions, as illustrated by the line 760, as compared to
incubating in buffer only, as illustrated by the line 770. FIG. 7A
highlights the release characteristics of biocompatible gels of
varying crosslinking densities on treatment with a non-sequence
specific endonuclease DNase I. FIG. 7B conclusively shows that a
DNase I trigger is responsible for the cleavage of DNA covalently
incorporated within a biocompatible gel.
Experimental Section
[0060] OBJECTIVE 1: The rational design, synthesis,
characterization, and optimization of novel biohybrid hydrogel
carriers with enhanced loading and intelligent triggered release.
The model therapeutic will be an anti-Human Immunodeficiency Virus
type 1 therapeutic (anti-HIV deoxy-ribozyme). The trigger will
involve physical mechanisms (e.g., temperature, pH changes) as well
as a specific biomolecular interaction, which will allow a rational
tailoring and control of the therapeutic release profile.
Specific Aims:
1) Analyze the critical factors for hybridization and restriction
enzyme digestion of the acrylated DNA helix;
2) Synthesize novel hydrogels with a covalently linked pendant DNA
helix via redox polymerization and within the gel;
3) Characterize and optimize loading efficiency of acrylated DNA
helix;
4) Analyze the in-vitro dynamic release of the cleaved DNA from the
network via temperature change, pH change, a sequence specific
enzymatic reaction;
5) Conduct diffusional analysis of cleaved DNA and restriction
enzyme through network;
6) Synthesize networks of varying macromolecular structures (e.g.,
crosslinking densities) and determine the release characteristics
of the cleaved DNA from biocompatible gels (e.g., synthesized from
FDA-approved monomers);
7) Demonstrate physiological relevance of system with dynamic
release studies of anti-HIV deoxyribozyme;
8) Perform network structural analysis via equilibrium weight and
volume swelling studies.
Experiments:
[0061] In-vitro Hybridization. The 5' acrydite oligonucleotide and
its complementary oligonucleotide are synthetically prepared and
resuspended to 1 mM in Tris buffer, 0.5 mM EDTA, pH 8. The
complementary and non-complementary oligonucleotides are
radiolabeled with ?-32P ATP using 3'phosphatase-free polynucleotide
kinase and purified on a denaturing 12% polyacrylamide gel.
Annealing of DNA strands to form double helical DNA is confirmed by
the dose-dependent addition (0, 100, 200, 300 and 500 picomoles) of
acrylated oligonucleotide to an aliquot of complementary
oligonucleotide (5000 cpm). Each pair of oligonucleotides are
heated to 90.degree. C. for 3 minutes and directly placed on ice.
Hybridization conditions are optimized in different buffers such as
water and various concentrations of Tris-HCl, pH 7.5. Samples are
analyzed by electrophoresis on 12%
poly(acrylamide-co-bisacrylamide) non-denaturing gel. Formation of
DNA duplexes are quantified using Typhoon phosphoimager and
ImageQuant software (Molecular Dynamics).
[0062] In-vitro Restriction Enzyme Digestion. Digestion of the DNA
helices are carried out by incubating the DNA helices with
restriction enzyme and restriction enzyme buffer at 37.degree. C.
for 1 hour, as per the manufacturer's instructions. Control
experiments are performed in restriction enzyme buffer only
(absence of BamHI) at 37.degree. C. for 1 hour, or on ice. Digests
are analyzed by electrophoresis on non-denaturing gel.
Autoradiograms are quantified using Typhoon phosphoimager and
ImageQuant software.
[0063] Synthesis and Characterization of Biocompatible DNA Gels.
Novel poly(acrylamide-co-N,N'methylenebisacrylamide-co-acrylated
DNA), poly(acrylamide-co-polyethylene glycol 200
dimethacrylate-co-acrylated DNA), poly(2-hydroxyethyl
methacrylate-co-polyethylene glycol 600 dimethacrylate-co-acrylated
DNA) hydrogels of varying crosslinking densities are prepared via
redox polymerization at 25.degree. C. in the loading lanes of a
non-denaturing gel. After polymerization, the unincorporated
acrylated DNA and unhybridized 32P-labeled oligonucleotides are
efficiently eluted by electrophoresis. The polymer gels in the
loading lanes are then cut out and submerged in 10 mM Tris buffer,
pH 7.5. The rest of the base gel is dried and imaged as described
earlier, in order to quantify the amount of DNA incorporated into
the DNA gels, and hence determine the efficiency of polymerization
reaction.
[0064] In-vitro Kinetic Release of DNA Strands upon Restriction
Enzyme Trigger. Release studies of 32P-labeled loaded DNA are
conducted by incubating the DNA gels under physiological conditions
in the presence of BamHI and BamHI buffer, and the release is
monitored by Cerenkov counting. Control experiments are performed,
by incubating the DNA gels in BamHI buffer (absence of BamHI), and
by using the restriction endonuclease EcoRI, which does not
recognize the BamHI recognition site. The enzymatic activity of
BamHI is lowered by decreasing the pH and release will be
monitored. Each experiment is performed five times and cumulative
and differential DNA released versus time, normalized dynamic
release profiles, and drug diffusion coefficient will be calculated
by using Fick's Law with a diffusion coefficient that is dependent
on position via one-dimensional planar solute release from the
gel.
[0065] In-vitro Kinetic Release of DNA Strands upon Deoxynuclease I
Trigger. Release studies of 32P-labeled loaded DNA are conducted by
incubating the DNA gels under physiological conditions in the
presence of DNase I (which is a non-specific endonuclease), and the
release is monitored by Cerenkov counting. Control experiments are
performed, by incubating the DNA gels in DNase I buffer (absence of
DNaseI). The effect of the macromolecular architecture the
diffusion of DNA strands from the gel to give tunable release
profiles are investigated by conducting release studies in gels of
varying crosslinking densities. Each experiment is performed five
times and cumulative and differential DNA released versus time,
normalized dynamic release profiles, and drug diffusion coefficient
will be calculated by using Fick's Law with a diffusion coefficient
that is dependent on position via one-dimensional planar solute
release from the gel.
[0066] In-vitro Kinetic Release of DNA Strands upon Temperature
Ramp Temperature-responsive release of melted DNA strands are
investigated between 30.degree. C. and 85.degree. C. Each
experiment is performed five times and cumulative and differential
DNA released versus time, normalized dynamic release profiles, and
drug diffusion coefficient will be calculated by using Fick's Law
with a diffusion coefficient that is dependent on position via
one-dimensional planar solute release from the gel.
[0067] Physiological validation of the platform: Downregulation of
HIV-1 Tat/Rev mRNA. The physiological relevance of the platform in
context of clinical medicine is demonstrated by following similar
steps to release an anti-HIV deozyribozyme. The double stranded
helix substrate is redesigned to include a catalytic unit of a DNA
enzyme that can bind to and cleave HIV-1 TAT/Rev mRNA, the coding
sequence for Tat and Rev proteins, along with the BamHI recognition
site. As Tat and Rev represent two essential proteins of human
immunodeficiency virus type 1 (HIV-1), mRNAs encoding these
proteins constitute frequent targets for DNA- and RNA-based gene
therapy. Treatment of the DNA gels with BamHI results in the
cleavage of the anti-HIV deozyribozyme from the gel, and
downregulation of the Tat mRNA by the mechanism stated above. The
HIV aptamer is synthesized in vitro using synthetic DNA templates
under the control of promoters specific to T7 RNA polymerase. The
aptamer is then radiolabeled using [5'-32P]pCp and T4 RNA ligase
and purified on a 12% poly(acrylamide-co-bisacrylamide)
non-denaturing gel. 3'-end labeled HIV aptamer is incubated along
with the DNA gels and BamHI under physiological conditions, and the
cleaved RNA fragments are analyzed by electrophoresis on a
denaturing gel. Autoradiograms are quantified using Typhoon
phosphoimager and ImageQuant software.
[0068] Physiological validation of the platform: Downregulation of
miRNA for Age-Related Macular Degeneration. The physiological
relevance of this platform in the context of developmental biology
and clinical intervention are demonstrated by following similar
steps to release an anti-miRNA ribozyme. The double stranded helix
substrate includes a siRNA construct which targets and interferes
with the functioning of the vascular endothelial growth factor
receptor (VEGFR). Treatment of the DNA gels with BamHI results in
the cleavage of the siRNA from the gel, and downregulation of the
VEGFR mRNA. The VEGFR mRNA is synthesized in vitro using synthetic
DNA templates under the control of promoters specific to T7 RNA
polymerase. The aptamer is then radiolabeled using [5'-32P]pCp and
T4 RNA ligase and purified on a 12%
poly(acrylamide-co-bisacrylamide) non-denaturing gel. 3'-end
labeled VEGFR aptamer is incubated along with the DNA gels and
BamHI under physiological conditions, and the cleaved RNA fragments
are analyzed by electrophoresis on a denaturing gel. Autoradiograms
are quantified using Typhoon phosphoimager and ImageQuant
software.
[0069] Kinetic Diffusion Studies in DNA Gels. Permeation studies
are conducted using Valia-Chen diffusion cells consisting of donor
and acceptor reservoirs with temperature control at 37.degree. C.
Each gel is pre-swollen in Tris buffer until equilibrium and placed
between the two half diffusion cells. The transport of restriction
endonuclease/cleaved fragments is determined via MALDI
spectrophotomer. Permeability coefficients, partition coefficients,
and diffusion coefficients are calculated. Comparison of the
relative rates of diffusion between the influx of BamHI and the
exodus of the cleaved fragments will yield information regarding
the rate limiting steps of the reactive/diffusion process.
[0070] Structural Analysis. Equilibrium weight and volume swelling
studies are conducted using conventional methods to calculate
polymer fractional content, gel structural properties such as
molecular weight between crosslinks, and correlation length (e.g.,
mesh size of the gel).
[0071] OBJECTIVE 2: The rational design, synthesis,
characterization, and optimization of novel biohybrid hydrogel
carriers which exhibit modulatory, on-off, release of a therapeutic
via reversible aptamer-analyte crosslinking.
Specific Aims:
1) Perform in-vitro transcription and purification of a
biotin-specific aptamer and characterize via equilibrium and
kinetic binding isotherms;
2) Synthesize novel recognitive RNA-based hydrogels using aptamer
and bioconjugate chemistry;
3) Elucidate the associated mesh size changes and swelling
transitions via metal ion/chelating agent switching;
4) Perform dynamic release kinetics of a fluorescent molecule from
the hydrogel carrier.
Experiments:
[0072] In-vitro Transcription and Purification of an Analyte
Specific Aptamer. Biotin-binding RNA pseudoknots are synthesized in
vitro using synthetic DNA templates under the control of promoters
specific to T7 RNA polymerase. Large quantities of DNA are
generated using the Klenow fragment of DNA Polymerase I. Integrity
of the transcripts are examined by denaturing polyacrylamide gel
electrophoresis. Biotin binding pseudoknots are optimized for
binding efficiency using SELEX. Pseudoknots are radiolabeled using
[5'-32P]pCp and T4 RNA ligase and purified on a 12%
poly(acrylamide-co-bisacrylamide) non-denaturing gel. Modified
nucleotides are co-transcriptionally incorporated into RNA
pseudoknots to render them resistant against ribonucleases and then
selected for binding via affinity chromatography. For example, by
modulating incorporation of fluorine-pyrimidine nucleotides
(Epicentre biotechnologies) small RNAs resilient against
degradation by ubiquitous RNAse A, can be produced. Protection from
other RNases is achieved by posttranscriptional modification of
accessible nucleotides with nucleotide specific reagents (kethoxal,
DMS, DEPC).
[0073] Affinity Elution Studies. Binding properties of RNA aptamers
are monitored using modified Sepharoses. Columns are equilibrated
with Biotin Binding Buffer (20 mM HEPES, 100 mM KCl, 15 mM MgCl2).
Loading the column with samples or magnesium/EDTA buffers is
followed by spinning at 1000 rpm for 20 seconds.
[0074] Synthesis of Novel Recognitive RNA based Hydrogels using
Aptamer and Bioconjugate Chemistry. Poly(acrylic
acid-g-RNA-g-biotin) networks are prepared from poly(acrylic acid)
chains of high monodispersity. Grafting of RNA and modified biotin
amines to the matrix occur via commercially available cross-linking
reagents (Maleimide chemistry-EDC, oxene chemistry). Hydrogels are
assembled in polystyrene molds of precise dimensions.
[0075] Elucidation of Mesh Size Changes/Swelling Transitions via
Metal Ion/Chelating Agent Switching. Observable macroscopic volume
transitions are induced by metal ion/chelating agent switching,
which are observed under an optical microscope. Gels are placed in
known of volume of Tris buffer and the concentration of a FTIC
Dextran (model drug) versus time at 37.degree. C. will be used to
determine modulatory release kinetics.
[0076] The present invention has been described in terms of
specific embodiments incorporating details to facilitate the
understanding of the principles of construction and operation of
the invention. Such references herein to specific embodiments and
details thereof are not intended to limit the scope of the claims
appended hereto. It will be apparent to those skilled in the art
that modifications can be made in the embodiments chosen for
illustration without departing from the spirit and scope of the
invention. Specifically, it will be apparent to one of ordinary
skill in the art that the device of the present invention can be
implemented in several different ways and the apparatus disclosed
above is only illustrative of the preferred embodiment of the
invention and is in no way a limitation.
* * * * *