U.S. patent application number 17/123808 was filed with the patent office on 2021-08-19 for magnetic nanoparticles functionalized with catechol, production and use thereof.
The applicant listed for this patent is COLOROBBIA ITALIA S.P.A.. Invention is credited to Giovanni BALDI, Marisa BENAGIANO, Marco BITOSSI, Mauro COMES FRANCHINI, Mario Milco D'ELIOS, Costanza RAVAGLI.
Application Number | 20210252171 17/123808 |
Document ID | / |
Family ID | 1000005550139 |
Filed Date | 2021-08-19 |
United States Patent
Application |
20210252171 |
Kind Code |
A1 |
BALDI; Giovanni ; et
al. |
August 19, 2021 |
MAGNETIC NANOPARTICLES FUNCTIONALIZED WITH CATECHOL, PRODUCTION AND
USE THEREOF
Abstract
There are described magnetic nanoparticles the surface of which
is functionalized with catechol and constructs comprising a
plurality of said nanoparticles encapsulated in a biocompatible
polymer matrix, wherein a molecule with therapeutic action is
optionally dispersed, said polymer matrix optionally being in turn
further functionalized; there are further described cells of the
immune system incorporating said polymeric constructs giving rise
to their engineering.
Inventors: |
BALDI; Giovanni; (Montelupo
Fiorentino, IT) ; RAVAGLI; Costanza; (Sesto
Fiorentino, IT) ; COMES FRANCHINI; Mauro; (San
Lazzaro Di Savena, IT) ; D'ELIOS; Mario Milco;
(Empoli, IT) ; BENAGIANO; Marisa; (Roma, IT)
; BITOSSI; Marco; (Montelupo Fiorentino, IT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
COLOROBBIA ITALIA S.P.A. |
Sovigliana Vinci |
|
IT |
|
|
Family ID: |
1000005550139 |
Appl. No.: |
17/123808 |
Filed: |
December 16, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15110189 |
Jul 7, 2016 |
10888630 |
|
|
PCT/IB2015/050122 |
Jan 7, 2015 |
|
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17123808 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 9/5031 20130101;
A61K 9/1075 20130101; A61K 49/186 20130101; A61K 9/5089 20130101;
A61K 49/1857 20130101; H01F 1/0063 20130101; A61K 41/0052 20130101;
H01F 1/0054 20130101; A61K 9/20 20130101 |
International
Class: |
A61K 49/18 20060101
A61K049/18; A61K 9/20 20060101 A61K009/20; A61K 41/00 20200101
A61K041/00; A61K 9/50 20060101 A61K009/50; H01F 1/00 20060101
H01F001/00; A61K 9/107 20060101 A61K009/107 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 7, 2014 |
IT |
FI2014A000003 |
Claims
1. (canceled)
2. A construct comprising a plurality of magnetic nanoparticles
consisting of nanometric magnetite particles whose surface is
functionalized with catechol encapsulated in a biocompatible
polymeric matrix wherein a molecule having therapeutic action is
optionally dispersed.
3. The construct according to claim 2, further comprising a
plurality of gold nanorods.
4. The construct according to claim 2 wherein said biocompatible
polymeric matrix consists of biodegradable copolymers.
5. The construct according to claim 4, wherein said biodegradable
copolymers are selected from: biodegradable nanomicelles,
polyesters, polyurethanes, polycarbonates and poly(glutamic) acid,
polyetheramine and polybenzylglutamate.
6. The construct according to claim 5, wherein said biodegradable
nanomicelles consist of poly(lactic-co-glycolic) acid and
polyethylene glycol carboxylate (PLGA-b-PEG-COOH), having formula
(I) ##STR00007## wherein m=[117-330]; n=[117-330]; p=[60-100].
7. The construct according to claim 2, wherein said molecules with
therapeutic action are selected from: anticancer agents,
peroxynitrite scavengers, superoxydismutase inhibitors, retinoids,
cytokines, aspirin.
8. The construct according to claim 6, wherein the end carboxyl
group of the fragment PEG-COOH of the micelles is further
functionalized with monoclonal antibodies, proteins, peptides or
active molecules of interest for the specific recognition by the
cellular over-expressions.
9. The construct according to claim 8, wherein said antibodies are
selected from: hEGR, hEGFR, IgG, moAb.
10. A process for preparing the construct according to claim 2,
wherein: an organic solution of polymer dissolved in a solvent,
mixed with the suspension of nanoparticles coated with organic
binder, both in the same solvent, and an aqueous solution of
Na2HPO4 1 mM) are mixed in a constant flow in a mixing cell with
batch or continuous synthesis.
11. A process for preparing the construct according to claim 10,
wherein the end carboxyl group of the fragment PEG-COOH is
activated so as to promote the subsequent attack by esterification
of amino end groups.
12-13. (canceled)
14. Use of the construct of claim 2 for hyperthermia
treatments.
15. Use of a human immune system cell modified to contain the
construct of claim 2 for the diagnostic of cancer, degenerative,
central nervous system, cerebral cardiovascular diseases,
infectious diseases, transplants, autoimmune diseases and also for
the treatment of tumors, cerebral cardiovascular diseases,
degenerative diseases (e.g. Alzheimer's), infectious diseases,
transplants, liver cirrhosis and other diseases characterized by
fibrogenesis, diseases characterized by recurrent fetal loss,
intrauterine fetal death, neonatal diseases, congenital and
acquired coagulation disorders, genetic disorders, autoimmune
diseases and, finally, for pain relieving.
16. Use of the construct of claim 2 as an MRI imaging means.
17. Use of the construct according to claim 3 for the diagnosis and
treatment of tumor diseases.
Description
[0001] This application is a continuation of U.S. patent
application Ser. No. 15/110,189, filed Jul. 7, 2016, which is a
National Stage Application under 35 U.S.C. .sctn. 371 of PCT
Application No. PCT/162015/050122, filed Jan. 7, 2015, which claims
priority benefit of Italy Patent Application No. FI2014A000003,
filed Jan. 7, 2014, which are hereby incorporated by reference in
their entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to the field of functionalized
nanoparticles, their production and their use.
PRIOR ART
[0003] As known, magnetite is a mineral with ferromagnetic
properties whose chemical formula is Fe.sub.3O.sub.4 (sometimes
also written as FeO.Fe.sub.2O.sub.3).
[0004] It is well known that magnetite in nanoparticle form, i.e.
with dimensions ranging from a few nanometers to a few tens, if
immersed in a variable magnetic field in the range of radio waves,
interacts with the electromagnetic field and then releases thermal
energy to what is around it, thus giving rise to what is called
hyperthermic effect or magnetic hyperthermia.
[0005] In oncology, hyperthermia is exploited to improve the
efficacy of chemotherapy or radiotherapy; in fact, raising the
temperature of a solid tumor between 41 and 45.degree. C. induces
the apoptosis of the tumor cells; generally, this is applied by
means of washings with liquids brought to the appropriate
temperatures and circulated in the vicinity of the sites affected
by tumor masses.
[0006] Recently, antennas are adopted which, inserted directly into
the tumor mass, generate microwaves and thus interact with the
dipole molecules of water, generating hyperthermia.
[0007] These treatments are generally extremely invasive and of
poor efficacy (in the first case) and not devoid of possible
negative side effects such as risk of metastasis, tissue necrosis,
etc. in the second case.
[0008] Using magnetic nanoparticles that arrive in the immediate
vicinity of the tumor tissues or preferably that penetrate into
cancer cells, it is possible to overcome the above problems and
achieve a high efficiency of the hyperthermic effect, localizing it
at the cellular level.
[0009] Specifically sending nanostructures in the tumor cells of
solid tumors, or on pathological tissues or sites such as
Alzheimer's amyloid plaques, or on the damaged tissues of multiple
sclerosis, it is thus possible to convey, in an efficient manner
and free from side effects, multiple conjugated treatments, such as
the hyperthermic and pharmacological ones.
[0010] In literature there are many examples of hybrid
inorganic-polymer or protein nanoparticles comprising a
biocompatible core of nanoparticle magnetite and a coating, either
polymer or protein, possibly loaded with drugs and functionalized
on the surface, with suitable targeting agents.
[0011] These nanoparticles are potential theranostic agents wherein
the ability to generate heat under the effect of an electromagnetic
EM field (hyperthermic effect), the possibility of drug delivery
(DD) and the ability to be identified during its action with
imaging techniques (MRI) are synergistically combined.
[0012] The International Patent Application WO 2004/071386
describes compounds consisting of mono- or bi-lamellar liposomal
microcapsules containing a magnetic nanoparticle and a biologically
active molecule having the primary aim to reach and treat liver
tumors.
[0013] In European patent EP 1 979 365, the Applicant has described
constructs consisting of nanometric magnetic particle
functionalized with bifunctional molecules wherein an end of said
molecules is bound to the surface of the magnetic particle while
the other is free and can therefore be reacted with complex units
such as biopolymers, cyclodextrins, antibodies and drugs for use in
the pharmaceutical and diagnostic field, allowing
nanoparticle/binder complexes to be obtained wherein there occurs a
total and compact coating of the nanoparticle without significant
alterations of the properties depending on it (e.g. optical or
magnetic properties).
[0014] The subsequent patent EP 2 117 600 describes constructs in
which the functionalized particles similar to those described in
the above patent EP 1 979 365 are coated with polymers in which a
molecule having pharmacological properties has possibly been
dispersed.
[0015] Also European Patent Application 2 512 992 (to the name of
the same Applicant) describes polyol synthesis processes which
allow easily obtaining magnetite nanoparticles with even and
controlled size (which therefore have a high hyperthermic
efficiency).
[0016] As can be seen, therefore, many solutions have been
suggested in the literature for the solution of the problem of
selectively directing within the body particles capable of
performing a therapeutic action both by application of hyperthermia
alone or in combination with traditional drugs; however, known
products do not fully meet the application needs to achieve an
effective treatment of tumors and other diseases with
nanostructures due to various problems not yet overcome.
[0017] The first problem is the specificity of the nanostructure,
in fact it is known from the literature that hybrid
inorganic-polymer/protein particles are quickly eliminated from the
reticuloendothelial system when administered systemically
(reticular cells, macrophages, Kupffer cells).
[0018] The clearance of the nanostructures is therefore responsible
for the inefficiency of a nanotheranostic treatment at a systemic
level; numerous attempts have been made to overcome this
difficulty, including the functionalization of the nanoparticle
polymer/protein surface with delivery units such as monoclonal
antibodies, peptides and active molecules (such as sugars, etc.)
but also in this case, most of the particles are eliminated by the
reticuloendothelial system and only a small amount reaches the
sites concerned, the tumor tissue and cancerous cells.
[0019] A second problem, a consequence of the first one, is that
the amount of magnetic particles that reach the tumor or the
pathological tissue may prove insufficient to carry out an
efficient hyperthermic effect.
[0020] Finally, the current nanotheranostic systems have poor
stability in biological fluids and thus tend to form large
aggregates (up to over 500-1000 nm) that are unlikely to penetrate
into the tumor mass or go beyond an intact blood-brain barrier,
which worsens the specific targeting of these systems in the target
cells, further limiting the efficiency of the treatment.
[0021] From the literature it is known that T lymphocytes within
the immune system are the main protagonists of the anti-tumor
responses.
[0022] They are able to selectively recognize the tumor cells due
to their specific receptor, called TCR. The T lymphocyte activation
by the respective tumor antigenic peptide may occur only if the
antigen is presented by the cells represented by monocytes,
macrophages, dendritic cells, Langerhans cells, microglia or also B
lymphocytes. For an effective T lymphocyte activation, membrane
signals and soluble signals are also required in addition to the
antigen. Among the soluble signals, the most powerful activation
factor is interleukin 2 (IL-2), while among the membrane signals,
the most powerful is molecule B7.
[0023] Once the tumor is identified, it is destroyed by the
lymphocytes through various mechanisms, among which the main ones
are: the cytotoxic machinery linked to perforin and that linked to
Fas ligand.
[0024] Melanoma was one of the first tumors to be associated with a
strong local immune response mediated by T lymphocytes, and over
the years it has been possible to prove that a strong T-Lymphocytic
response is related to a better prognosis.
[0025] Through the use of nanoparticles, it is possible to develop
a new custom anticancer strategy based on the use of T lymphocytes
specialized in killing a tumor, armed by nanoparticles, ready to
hit the tumor, after activation by laser/electromagnetic
fields.
[0026] Moreover, the literature widely describes the role played by
the immune system, and in particular by lymphocytes and
inflammatory cells, in diseases of the nervous system such as
multiple sclerosis, Alzheimer's disease.
[0027] Multiple sclerosis is indeed the prototype of autoimmune
diseases in the pathogenesis of which a crucial role is played by T
lymphocytes (Elliot M. Frohman, M. D., Michael K. Racke, M. D., and
Cedric S. Raine, N Engl J Med 2006; 354:942-955). In particular,
T-helper cells capable of producing important inflammatory
cytokines, such as interferon-gamma and lymphotoxin, called
T-helper cells 1 (Th1), but also T lymphocytes CD8, B lymphocytes
and immune cells of the monocyte line, are very important. Also in
Alzheimer's disease (Henry W. Querfurth, and Frank M. LaFerla, N
Engl J Med 2010; 362:329-344), an important pathogenetic role is
carried out by inflammatory mechanisms related to the production of
interleukin 1, interleukin 6, tumor necrosis factor .alpha., by
microglia and astrocytes, due to amyloid proteins; nanoparticles
according to the invention can therefore play an important role
also in the treatment of these diseases.
BRIEF DESCRIPTION OF THE FIGURES
[0028] FIG. 1 shows, taken with a Field emission gun scanning
microscope in STEM mode, the typical cluster formation taken by the
nanoparticles according to the invention within a polymer
matrix.
[0029] FIG. 2 shows a mixture construct of magnetic particles and
gold nanorods.
[0030] FIG. 3 schematically shows a construct model consisting of
nanoparticles of magnetite or magnetite and gold nanorods and
coated with block polymers PLGA-b-PEG-COOH.
[0031] FIG. 4 shows the layout of the step by step preparation
process of the nanostructured construct according to the
invention.
[0032] FIG. 5 shows the layout of the process for the purification
and selection of lymphocytes.
[0033] FIG. 6 shows a picture taken with an optical microscope of
monocytes/macrophages charged with nanoparticles according to the
invention.
[0034] FIGS. 7 and 8 show the 1H-NMR of the polymer PLGA-NHS
conjugated with NH2-PEG-COOH.
[0035] FIG. 9 shows the UV-Vis spectrum of a product according to
the invention.
[0036] FIG. 10 shows a BCA Test on a product according to the
invention.
SUMMARY OF THE INVENTION
[0037] There are described magnetic nanoparticles the surface of
which is functionalized with catechol and constructs comprising a
plurality of said nanoparticles encapsulated in a biocompatible
polymer matrix, wherein a molecule with therapeutic action is
optionally dispersed, said polymer matrix optionally being in turn
further functionalized. It was surprisingly found that said
polymeric constructs can be incorporated into immune system cells
giving rise to the engineering thereof.
DETAILED DESCRIPTION OF THE INVENTION
[0038] It has now been surprisingly found that constructs
comprising a plurality of magnetic nanoparticles functionalized
with catechol encapsulated in a biocompatible polymer matrix can
overcome the above problems, ensuring the necessary stability in
physiological media and in human blood.
[0039] Moreover, the structural features of these constructs helps
ensure an implemented hyperthermic effect compared to that shown by
monodisperse inorganic cores described in the above patents; this
advantage is due to a so-called "cluster structure" (see FIG. 1) of
the magnetic particles which tend to combine in structural centers
of multiple particles within the polymer matrix carrying out a
synergistic effect on the hyperthermic properties.
[0040] The functionalization of the magnetic particles with
catechol, according to the invention, is essential for the above
cluster structure to occur and therefore allows obtaining much
superior constructs than those known in the prior art as regards
hyperthermic properties and stability over time.
[0041] Among the magnetic particles, magnetite is especially
preferred. If preferred, the constructs according to the invention
may have, in addition to the magnetic nanoparticles as described
above, a plurality of gold nanorods (see FIG. 2).
[0042] The presence of nanorods allows a considerable hyperthermic
effect by applying an infrared laser radiation such as that
generated by CO2 lasers, which goes to further increase the
hyperthermic effect imparted by cluster structures of
magnetite.
[0043] This enables a combined laser and radio waves system which
uses laser for surface districts or those that can be reached via
probe and the radio waves for deep districts. Magnetic
nanoparticles can be prepared through the known polyol process as
described for example in the above European patent application 2
512 992 which describes a preparation process in which:
i) a polyol solution of Fe.sup.III is prepared starting from
Fe.sup.0; ii) magnetite nanoparticles are prepared in the polyol
synthesis conditions.
[0044] The above step (i) is the well-known and described reaction
of acid attack (also weak acids such as acetic acid) on iron
according to the equation:
Fe.sup.0+2H.sup.+Fe.sup.2++1/2H.sub.2.uparw.
[0045] Thereafter, it is possible to completely oxidize the
solution of Fe.sup.II in polyols to Fe.sup.III (for example
acetate) through air flushing and addition of H.sub.2O.sub.2 in the
reaction environment at a temperature of less than 100.degree.
C.
[0046] Gold nanorods are prepared in known manner with a
microwave-assisted synthesis starting from gold in ionic form in
the presence of various additives: alkyltrimethylammonium bromide,
CnTAB n=10-16, cetylpyridinium chloride, C16 PC and PVP [in this
regard, see M. Tsuji, K. Matsumoto, T. Tsuji, H. Kawazumid, Mater.
Lett. 59 (2005) 3856] or by reduction of HAuCl.sub.4 with ascorbic
acid in the presence of CTAB and AgNO3 (in this regards, see Ratto
F. et al. J NANOPARTICLE RESEARCH 2010 and [Ratto F. et al. J
NANOPARTICLE RESEARCH 2012)
[0047] The surfaces of the magnetic and/or magneto-optical
particles obtained as described above are functionalized with
catechol (bifunctional group) by exploiting the affinity of the
polar groups OH to the surface of the particles and allowing the
end part not bond to the particle surface to maintain a hydrophobic
reactivity suitable for the subsequent incorporation in a
polymer/protein matrix.
[0048] The polymer matrix according to the present invention is
understood to consist of biodegradable copolymers and is thus
capable of allowing the release of the drug, which must proceed
gradually as the matrix degrades in a physiological
environment.
[0049] Examples of suitable copolymers for the purpose are:
biodegradable nanomicelles, polyesters, polyesters, polyurethanes,
polycarbonates and poly(glutamic) acid, polyetheramine and
polybenzylglutamate.
[0050] Particularly preferred are biodegradable nanomicelles,
consisting of block copolymers of poly(lactic-co-glycolic) acid and
polyethylene glycol carboxylate (PLGA-b-PEG-COOH, MnPLGA
range=44-10 kDa, MnPEG=2-3 kDa) having formula (I)
##STR00001##
wherein m=[117-330]; n=[117-330]; p=[60-100].
[0051] This product is known and has already been employed in
various other works of Drug Delivery also at the level of Clinical
Phase I for testing of anticancer agents (see X. Shuai et al, 2004
and X. Shuai, H. Ai, N. Nasonkla, S. Kim, J. Geo, J. Controlled
Release, 2004, 98, 415).
[0052] The polymer in fact has features that allow assembling
nanospherical systems with a hydrophobic inner area, guaranteed by
residues of PLGA, and a hydrophilic outer area which is imparted by
the terminals of PEG-COOH (see FIG. 3).
[0053] This dual feature allows the nanospheres to trap the organic
active ingredients in the hydrophobic part and to be dispersed in
aqueous solution thanks to the hydrophilic part. If desired, the
polymer can be admixed with molecules having a therapeutic action
which is dispersed in the polymeric matrix according to known
processes and as illustrated in the examples given below.
[0054] Examples of molecules with therapeutic action according to
the invention are for example anti-cancer drugs (taxanes,
gemcitabine, vincristine, etc.), peroxynitrite scavengers,
superoxide dismutase inhibitors, retinoids (bexarotene), cytokines
such as interleukin 10, TLR-ligands such as the HP-NAP molecule
capable of activating TLR2, aspirin.
[0055] In addition, the carboxylic acid functionality of the
PEG-COOH fragment of the micelles allows a chemical stable bond
with monoclonal antibodies, proteins, peptides or active molecules
of interest (for example, and/or fluorescent dyes) for the specific
recognition by the cellular over-expressions.
[0056] Among the antibodies useful for the functionalization
according to the invention we may mention hERG, hEGFR, IgG, moAb,
etc.
[0057] The examples (see example 10) describe the above
functionalization, in particular using a specific monoclonal
antibody hERG1 described and claimed in Italian patent IT
1,367,861.
[0058] In particular, it is a specific monoclonal antibody against
the extra-cellular portion S5-pore of protein HERG1 produced by a
hybridoma comprising the product of a fusion between an
immortalized cell, belonging to the murine neoplastic cell line
NSO, and a lymphocyte obtained by immunization of a mouse with a
peptide of sequence EQPHMDSRIGWLHN.
[0059] The construct according to the invention (hereinafter also
referred to as "nanobioreactor" or "NBR") containing magnetic
nanoparticles functionalized with catechol is prepared by carrying
out a nanoprecipitation, wherein two fluids: [0060] an organic
solution of polymer dissolved in a solvent, mixed with the
suspension of nanoparticles coated with organic binder, both in the
same solvent, and [0061] an aqueous solution of Na2HPO4 1 mM) are
mixed in a constant flow in a mixing cell with batch or continuous
synthesis.
[0062] For the batch synthesis, the organic suspension containing
polymer and particles is injected with a syringe in the aqueous
solution, without magnetic stirring, in a single step.
[0063] For the continuous synthesis, a double peristaltic pump
system is prepared to carry out the addition of the organic
solution to the aqueous stream (organic volume/water ratio 1/10).
The respective tubes draw the solution directly from the lungs
containing the organic suspension (with functionalized particles
and polymer) and the solution of Na2HPO.sub.3 1 mM (pH 7.4).
[0064] Once the dispersion of hybrid particles (consisting of
magnetic nanoparticles functionalized with catechol included in the
polymer) has been obtained, part of the organic solvent is removed
via a rotary evaporator so as to minimize the amount of organic
phase in the subsequent production steps.
[0065] The suspension is then dialyzed against aqueous solution
Na2HPO3 for the removal of the organic phase and concentrated to
the minimum volume possible to obtain a concentration of from 0.1
to 1% w/w.
[0066] Through a second concentration it is possible to obtain a
much more concentrated product through membrane dialysis with a
theoretical concentration factor ranging from 5.times. to 20.times.
depending on usage. The product is then filtered with a filter to
0.22 .mu.m to remove the bacterial load. The product has an
excellent hyperthermic efficiency if irradiated for 30 minutes with
alternating magnetic field of 21-24 kA/m and a frequency of 160-190
kHz, its temperature increases by at least 5.degree. C.
[0067] The method described herein allows the preparation of
constructs with a dimensional distribution centered in a range from
30 to 60 nm.
[0068] The potential .xi. of the product thus obtained (Malvern
Zetasizer nano-S), measured to have information about the
electrostatic stability of the suspension and its ionic strength,
is lower than -30 mV, which means that the particles are affected
by the negative surface electrostatic repulsion produced by the
carboxylic groups which at (physiological) pH 7.4 are partially
deprotonated.
[0069] The experimental conditions described above allow making a
suspension with good stability after dilution into culture media
typically used for cell cultures (DMEM, RPMI), exhibiting little
tendency to aggregation and sedimentation also after a clear change
in ionic strength conditions due to dilution.
[0070] Obtaining the product functionalized on the surface with
monoclonal antibodies and/or fluorescent dyes (e.g. Cyanine.RTM.,
Dylight.RTM., etc.) for a targeted delivery and for use in imaging
techniques requires the use of NBR as a precursor before it is
subjected to the second concentration process (see above).
[0071] Typically, at this stage, the product has a concentration of
about 0.05-0.1% wt. of inorganic material.
[0072] The preliminary process step provides the activation of the
end carboxyl groups of the polymer, exposed towards the outer part
of the nanoparticle, in contact with the polar phase, with
activators such as EDAC [1-ethyl-3-(-3-dimethylaminopropyl)
carbodiimide hydrochloride] (molar ratio EDAC/COOH=10/1) and
sulfo-NHS (NHS/COOH=1/1), so as to promote the subsequent attack by
esterification of the end amine groups of the monoclonal antibody
and/or of the fluorescent dye.
[0073] In the case of fluorescent dyes with emission at
.lamda.600-800 nm (suitable for NIR imaging applications in vivo),
since only NHS ester-terminal molecules are available on the
market, it is necessary to provide for an intermediate step where a
diamino-terminal linker is added for the bridge link on the one
hand with the fluorescent dye, and on the other with the carboxylic
groups of the activated polymer.
[0074] Once the surface of nanoparticles has been activated, the
antibody and/or amino-terminal dye solution is added and let
stand.
[0075] The suspension is then concentrated and dialyzed against
aqueous Na.sub.2HPO.sub.3 and concentrated up to 0.2-1.0% wt. of
inorganic phase, depending on usage.
[0076] The product is then filtered with a filter to 0.22 .mu.m to
remove the bacterial load.
[0077] The method described herein allows the preparation of
constructs with a dimensional distribution centered in a range from
40 to 70 nm.
[0078] The potential .xi. of the product thus obtained (Malvern
Zetasizer nano-S), measured to have information about the
electrostatic stability of the suspension and its ionic strength,
is less than -30 mV, but greater than that measured on the NBR
product, which means that the negative charge exerted by the
carboxylic groups of the raw product is partly neutralized by the
bound antibody/dye.
[0079] The contents of antibody bound to the particles is analyzed
using the BCA.RTM. test: following the addition of suitable
reagents to the solution containing the protein analyte, a complex
of Cu.sup.2+ develops whose coloring at 562 nm is observed with
spectral analysis and from which the concentration of antibody is
derived using a linear calibration.
[0080] With the procedure described herein it is possible for
example to produce nanoparticles functionalized with moAb, with
moAb attack percentage between 5 and 30% wt compared to the
inorganic phase content.
[0081] The production of the nanobioreactor/lipophilic drug
(hereafter NBR_PTX) and nanobioreactor/antibody/lipophilic drug
(NBR_hERG_PTX) system (where the lipophilic drug for example is
Paclitaxel) is exactly the same as the synthesis process of the
nanobioreactor as described above and therefore provides for the
encapsulation of the inorganic nanoparticles, previously
functionalized with catechol, within a polymeric matrix based on
PLGA-b-PEG-COOH. The only variation to this process provides for
the dissolution of the specific amount of drug within the polymer
and the suspension of the functionalized nanoparticles.
[0082] Then, the nanobioreactor loaded with paclitaxel (NBR_PTX) is
obtained using the nanoprecipitation method, where the
aforementioned organic solution is vigorously added to the aqueous
solution of Na.sub.2HPO.sub.4 1 mm inside a mixing cell. There are
no changes in the morphological properties of the suspension from a
batch synthesis to a continuous one. The purification, filtration
and concentration processes applied are the same as described
above.
[0083] For the product characterization, in addition to the
determination of the average particle diameter, their potential
.xi. and the concentration of inorganic phase, the amount of active
ingredient encapsulated is also determined by high performance
liquid chromatography.
[0084] The product thus obtained and characterized can then be
further functionalized on the surface with targeting units such as
(hEGR, hEGFR, IgG, . . . ).
[0085] The process set up to this end accurately follows the
targeting procedure of the nanobioreactor NBR_moAb as described
above.
[0086] In fact, it provides for a preliminary step of activation of
the carboxyl groups present on the polymer, with activators such as
EDAC and sulfo-NHS and a step of reaction with the monoclonal
antibody, all according to the same proportions as set out in the
process previously described for the NBR_moAb.
[0087] The usual purification and characterization processes are
then performed. The nanoparticle suspensions thus obtained are
characterized by a mean hydrodynamic diameter of between 45 and 55
nm, while the potential .xi. is well below -30 mV.
[0088] According to a further embodiment of the invention, the
constructs as defined above, as an alternative to the decoration
with proteins or with antibodies, may be incorporated into cells of
the immune system.
[0089] Surprisingly, the constructs comprising clusters of
magnetite particles functionalized with catechol and coated with
block copolymers of poly(lactic-co-glycolic) acid and polyethylene
glycol carboxylate (PLGA-b-PEG-COOH, MnPLGA range=44-10 kDa,
MnPEG=2-3 kDa) as described above are easily incorporated by cells
of the immune system without compromising their functionality and
vitality.
[0090] Once the immune system cells are engineered with the
introduction of the constructs according to the invention, these
can be used for the diagnosis of tumor diseases, degenerative
diseases (e.g. Alzheimer's disease), of the central nervous system,
cerebral cardiovascular and infectious diseases, transplants,
autoimmune diseases and also for the therapy of tumors, cerebral
cardiovascular diseases, degenerative diseases (e.g. Alzheimer's
disease), infectious diseases, transplants, liver cirrhosis and
other conditions involving fibrogenesis, diseases characterized by
multiple abortions, intrauterine fetal death, neonatal diseases,
congenital and acquired coagulation disorders, genetic diseases,
autoimmune diseases, and finally for pain relief.
[0091] The induction of the release can take place with different
methods, such as the specific antigen (e.g. MAGE-3 in the case of
treatment of melanoma, MOG or myelin antigens in the treatment of
multiple sclerosis, etc.) or with appropriate immunomodulatory
substances such as IL-2, CD40 ligand, TLR-agonists, liposomes,
immunostimulating complexes (ISCOMS).
[0092] It should be noted, in fact, that an important property of
the cells of the immune system is represented by their ability to
reach almost all the districts of the body, therefore, their use as
a carrier to reach specific districts, carrying through the
construct according to the invention the particular product
required to the destination, exceeds the great current limitation
of the nanotheranostics represented by the low specificity of the
treatment. The cells of the immune system useful for the above
purpose are for example selected from:
[0093] T-lymphocytes, monocytes, macrophages, dendritic cells,
natural killer cells, B-lymphocytes, neutrophil granulocytes,
eosinophil granulocytes, basophil granulocytes, gamma delta
cells.
[0094] The cells are taken from the single patient, loaded with the
desired nanoparticles and then re-introduced in the same patient
topically or systemically.
[0095] The cells of the immune system will then be purified, as
described below, and in order to facilitate the
selective/preferential targeting of the body districts affected by
the disease in question, the cells can be treated ex vivo with
relevant antigens (or allergens), immunomodulatory drugs or
engineered with immuno potentiating or immuno suppressive
molecules.
[0096] One of the ways to select T cells for diagnostic or
therapeutic purposes is to enrich the number of T lymphocytes
specific for a particular antigen which can be a tumor antigen as
described above.
[0097] Lymphocytes, properly engineered with the constructs of the
invention, can, once in place, release the particles by means of
suitable chemical stimuli, the particles can then under irradiation
of electromagnetic fields in the range of radio waves exert
hyperthermia or release active ingredients such as antitumor drugs,
scavengers of molecules active in the oxidative stress of brain
tissues, anti-inflammatory molecules, etc. Nanoparticles can still
perform their functions even if they remain confined within the
lymphocytes themselves.
[0098] The magnetic nanoparticles can also perform the MRI imaging
function, being excellent T2 T2* contrast media (see the above
patents), nanoparticles containing gold nanorods may be used in
laser-mediated antitumor therapy and identified by methods of the
photoacoustic spectrometry type.
Purification and Selection of Lymphocytes
[0099] T lymphocytes for use against tumors are purified from the
peripheral blood or from the tumor site or from the patient's lymph
nodes after prior administration of the relevant tumor antigens, or
from other districts of the body as deemed relevant, using
standardized methods and/or with the aid of selective MACS.RTM.
methods (Current Protocols in Immunology 2013; D'Elios et al J
Immunol 1997; 158:962-967).
[0100] In order to select T lymphocytes specific for the tumor, T
lymphocytes are placed in culture with the relevant tumor antigen
(e.g. MAGE-3 for melanoma, at a concentration of 10 .mu.g/ml) in
complete RPMI medium for five days. Then, recombinant human IL-2 is
added every three days, and then the cells will be loaded with
nanoparticles, washed and then administered to the patient
topically and/or systemically.
[0101] T cells for use as diagnostic product, for example for
multiple sclerosis with magnetic resonance technology, are selected
for their specificity for myelin antigens or MOG (10 .mu.g/ml) or
other antigens as preferentially capable of achieving the
structures of the CNS. To this end, they are cultured with one or
more antigens for five days, then expanded with IL-2, and then
loaded with NP.
[0102] The same procedure can be used for other neurological
diseases, such as Alzheimer's disease, Parkinson's disease, stroke
and other cerebro-cardiovascular diseases using appropriate
relevant antigens.
[0103] Dendritic cells (highly efficient for their ability to
present the antigen to T lymphocytes, and thus greatly able to
activate T-lymphocytes) are obtained using traditional standardized
methods and/or with the aid of selective methods MACS.RTM. (Current
Protocols in Immunology 2013; Codolo et al. Arthr Rheum 2008;
58:3609-17). They will be incubated for 36-44 hours with the
desired antigen, then loaded with NP, washed and reinfused to the
patient for therapeutic or diagnostic purposes (see the process
layout in FIG. 5).
[0104] Natural killer cells and/or gamma delta lymphocytes, with
strong cytotoxic activity, are obtained using traditional
standardized methods and/or with the aid of selective MACS.RTM.
methods (Current Protocols in Immunology, 2013), they are then
loaded with the desired NP as well as possibly with other
immunomodulatory compounds, washed and reintroduced into the
patient for therapeutic (e.g. antitumor) or also diagnostic
purposes. The neutrophil granulocytes are obtained using
traditional standardized methods and/or with the aid of selective
MACS.RTM. methods (Current Protocols in Immunology, 2013), they are
then loaded with the desired NP as well as possibly with other
immunomodulatory compounds, washed and reintroduced into the
patient for diagnostic (e.g. to identify the presence of any foci
of infection in the body which cannot be identified by other
techniques) or also therapeutic purposes.
[0105] Also other cell types may be selected for diagnostic and/or
therapeutic use (such as effector cells to be used for the therapy
of tumors, autoimmune diseases, infections, degenerative diseases),
such as B lymphocytes, eosinophils, basophils, which are obtained
using traditional standardized methods and/or with the aid of
selective MACS.RTM. methods (Current Protocols in Immunology
2013).
[0106] They are then loaded with the desired NP or possibly with
other immunomodulatory compounds, washed and re-introduced into the
patient.
[0107] Immune cells loaded with nanoparticles can be used to
display with appropriate imaging techniques body districts that are
a location of the disease.
[0108] T cells and Jurkat cells are optimally filled with NP after
4 hours.
[0109] Monocytes/macrophages, dendritic cells, J774A.1 cells are
capable of incorporating the nanoparticles with a method according
to the invention in which monocytes/macrophages, dendritic cells,
J774A.1 cells are loaded with the nanoparticles (NP) at a
concentration of 0.05% in a suitable specific culture medium
(mmedium). To form the mmedium containing NP, the NP are first
dispensed and then the specific culture medium.
[0110] The mmedium consists of:
COMPLETE DMEM 10% FBS
COMPLETE DMEM:
[0111] DMEM HIGH Glucose (DME/HIGH). (EUROCLONE) (code: ECB7501L)
[0112] L-GLUTAMINE, solution 200 mM (100.times.). (EUROCLONE)
(code: ECB 3000D) [0113] PENICILLIN-STREPTOMYCIN Solution
(100.times.). (ATCC) (code: 30-2300) [0114] 10% fetal bovine serum
FBS: Fetal Bovine Serum, Qualified. (Sigma-Aldrich) (code:
F6178-100 mL)
[0115] Where necessary, autologous serum of the patient or media in
the absence of serum will be used instead of fetal bovine
serum.
[0116] Monocytes/macrophages, dendritic cells, J774A.1 cells are
optimally filled with NP after 2 hours but the incorporation
phenomenon is active after 15' up to 24 h.
[0117] FIG. 6 shows a picture taken with an optical microscope of
the monocytes/macrophages charged with nanoparticles.
[0118] The invention will be more and better understood in the
light of the examples given below, also noting FIGS. 4 and 5 which
schematically summarize the various steps for the preparation of
the construct and the engineering of the cells of the immune
system.
Example 1
Preparation of Iron Acetate in Diethylene Glycol DEG
Reagents:
[0119] 40 g Fe (Fe<99%<212 mm) equal to 0.716 mol; 800 g
water; 800 g CH3COOH (80%) equal to 10.67 mol; 46.64 g oxygenated
water (30%) equal to 0.41 mol; 0.12 g concentrated HCl; DEG
(diethylene glycol) 3850 g.
Synthesis:
[0120] Iron, the acetic acid and water solution and the
hydrochloric acid were loaded to a 5000 mL 4-necked flask under
nitrogen flow and the temperature was brought to 90.degree. C. and
maintained for 6 hours. The system was left to cool under N2 and
the solution was filtered to remove the undissolved Fe. The
oxygenated water is added dropwise to the clear solution placed in
a flask using a dripper, keeping the temperature at 35.degree. C.
for 1h, obtaining a clear solution equal to 1628.3 g having an iron
titer of 2.40% w/w. Excess acids are then stripped by a first
vacuum distillation at the T of 40.degree., a recirculation of the
dry part with water and removal by distillation two times (two
consecutive washes) and a final stripping at the T of about
50.degree.. 3850 g DEG are added to the dry so as to bring the
theoretical iron titer to the value of 1.01% w/w Fe.
Example 2
Preparation of Fe3O4 Nanoparticles in Diethylene Glycol
Reagents:
[0121] 1.50 g Fe.sup.0 (Fe.sup.0<99%, <212 mm) Fe.sup.0=0.179
mol; 150 g DEG; 1.2 g solution in DEG 1/10 HCl conc. 37%; 300.00 g
FeAc3 in DEG (1.01% w/w Fe.sup.III).
[0122] The metal iron and DEG were placed in a 500 mL 4-necked
flask under N.sub.2 and the temperature was brought to 150.degree.
C. The solution in DEG of hydrochloric acid was added to the system
and left under stirring for 5 minutes. Iron acetate is then added
in 10 equivalent aliquots, using a syringe, so as to ensure the
correct growth of the particles, bringing the temperature to
170.degree. C., the reaction ends within 24-36 hours.
[0123] The product was left to cool to 60.degree. C. and decanted
in a beaker, magnetically retaining the unreacted metal iron and
then filtered on a 0.45 .mu.m glass fiber.
[0124] 450 g of a nanosuspension of magnetite in diethylene glycol
having a titer in ionic Fe equal to 0.91%.+-.0.05, which expressed
in Fe.sub.3O.sub.4 corresponds to 1.253%.+-.0.05. Hyperthermia was
measured on this sample and the values were as shown in the
table
TABLE-US-00001 Field Frequency Starting Sample KA/m KHz T .degree.
(C.) SAR.sub.M Filtered Fe3O4 24 168 29.4 350.0 SAR.sub.M: Specific
absorption rate expressed on the mass of metal (Fe)
Example 3
Preparation of Organic Binder N-(3,4Dihydroxyphenethyl)Dodecanamide
(DDA)
##STR00002##
[0125] MW=335.48 g/mol
Reagents:
[0126] 25 g Dopamine hydrochloride equal to 0.1318 mol; 1 L THF; 45
mL Triethylamine 0.32 mol; 31.20 mL Lauroyl chloride 0.135 mol;
Purification and Crystallization
[0127] 400 mL THF (Aldrich 401757-2L-Lot STBC4923V)
[0128] 935 mL ethyl acetate (Aldrich 34972-2,5L-Lot 57BC011AV)
[0129] 315 mL petroleum ether (Aldrich 77379-2,5L-Lot
BCBG7367V)
Synthesis:
[0130] To a 5L 4-necked flask, dopamine hydrochloride and then THF
(1L) are placed under nitrogen atmosphere and then the
triethylamine is added, and the system is kept under stirring for
about 20' to obtain a white suspension.
[0131] To a 3 L flask with a flat bottom, THF (1L) and the
acylating agent are added. The solution is stirred and added to the
reagents contained in the 5 L flask using a peristaltic pump at a
rate of approximately 2 mL/min over 9h, obtaining a solution of
yellow-orange color with some white solid on the bottom.
Purification:
[0132] The organic phase that contains the synthesis product is
then purified and the latter is recovered from the by-product
formed during the reaction. The purification is carried out through
the removal of the solvent via rotavapor with two recirculation's
(2.times.200 mL). On the other hand on the solid residue, and on
the residual traces in the synthesis flasks, aqueous extraction and
treatment with ethyl acetate are carried out in a separating
funnel. The organic phases are all combined, dried with Na2SO4 and
finally brought to dryness in a rotavapor. 57 g of orange oily
product are obtained.
Crystallization:
[0133] 450 mL of a mixture of petroleum ether:ethyl acetate=7:3 are
added to the product. The suspension was put under cold water and a
white solid began to crystallize. The system was left 1 day to
rest.
[0134] The solid was filtered on a Buckner, washed with mother
liquor and dried using an oil pump. About 39 g were obtained (44 g
theoretical--yield 88.6%).
[0135] The mother liquor resulting from crystallization (2.45
g--orange brown solid) and from the washes were brought to dryness
(PRIME 27.13 g--dark brown solid).
Example 4
[0136] Surface functionalization of Fe3O4 nanoparticles (in
THF):
Reagents:
[0137] 40.0 g Fe.sub.3O.sub.4 dispersion equal to 2.16410.sup.-3
mol; 1089 mg DAA equal to 3.24710.sup.-3 mol; 120 mL EtOH; 80.0 g
THF.
[0138] 1089 mg DDA in 120 mL EtOH are solubilized in a 250 mL
flask; the solution thus prepared is added to magnetite with a 60
ml syringe. It is then sonicated for 1h in an ultrasound bath. The
sample is left to stand for a few minutes and then 60 mL H2O are
added and the NP are settled on neodymium magnet; the supernatant
is separated and nanoparticles are dispersed again in 80.0 g THF. 4
drops of triethylamine are added to the dispersion (the particles
disperse after about ten minutes).
Characterization
DLS
TABLE-US-00002 [0139] SAMPLE PDI Z-ave Dv1 % V1 Fe3O4-DDA 0.142
34.9 (.+-.0.4) 27.1 (.+-.0.5) 100
Example 5
[0140] Surface functionalization of Fe3O4 nanoparticles (in
acetone):
Reagents:
[0141] 4.0 g Fe3O4 dispersion equal to 0.2 0.10.sup.-3 mol; 108.0
mg DAA equal to 0.3 0.10.sup.-3 mol; 12.0 mL EtOH; 13.6 mL
acetone.
[0142] The suspension of magnetite is sonicated in an ultrasonic
bath for 1 h, then it is added to a solution of 108 mg DDA in 12.0
mL EtOH with a 25 mL syringe. Then, it is placed to sonicate for 30
min. The specimen is left to rest for a few minutes. 6 mL H.sub.2O
are added and NP are settled on neodymium magnet, then the
supernatant is separated and the NP dispersed again in 13.6 mL
acetone. 2 drops of triethylamine are added to the dispersion (the
particles disperse immediately).
Characterization
DLS
TABLE-US-00003 [0143] SAMPLE PDI Z-ave Dv1 % V1 Fe3O4-DDA 0.218
34.5 (.+-.0.2) 22.4 (.+-.0.5) 100
Example 6
Synthesis of Polymer PLGA-b-PEG-COOH 43-3 kDa
[0144] For the synthesis of the block copolymer PLGA-b-PEG-COOH,
the precursor PLGA-COOH (MW 44-10 kDa) was activated with
N-hydroxysuccinimide (NHS) using the coupling chemistry of
dicyclohexylcarbodiimide (DCC), and then combining the adduct with
the amino-functional part PEG-NH2 (MW 2-3 kDa) in dichloromethane
(DCM) as described hereinafter:
Step 1: Activation of the Carboxylic Functionality with NHS
##STR00003##
TABLE-US-00004 Group 1.037 g NHS (N-hydroxysuccinimide 98%) 720 mL
DCM (Dichloromethane >99.9%) 1.98 g DCC (N,N
Dicyclohexylcarbodiimide 99%) 990 mL DCM (Dichloromethane
>99.9%) 1600 mL Diethyl ether (.gtoreq.99.8)
Process
[0145] PLGA-COOH and 600 mL dichloromethane were added to a 5L
four-necked flask under nitrogen. After solubilization of the
polymer, N-hydroxysuccinimide (NHS) and then N,N
Dicyclohexylcarbodiimide (DCC- about 0.25 g at a time) were added;
the system was left under stirring for about 40h in an inert
atmosphere. 120 mL dichloromethane were used to wash the funnel
from the solids in order not to lose the raw materials.
[0146] The yellow suspension was filtered into a 2L tailed flask in
order to remove dicyclohexylurea. The 5 L flask was washed with 250
mL (.times.3) and 190 ml CH.sub.2Cl.sub.2.
[0147] The product was concentrated to approximately 400 mL volume
by a rotavapor in a 1 L flask (50 ml dry DCM wash): a dense
yellowish suspension was obtained.
[0148] The PLGA-NHS was precipitated using 400 mL (.times.4) of
cold diethyl ether. For each wash, the white solid was decanted and
the supernatant was removed. Subsequently, the solid is dried for
about 2h30' using the oil pump.
Step 2: Conjugation of PLGA-NHS with NH2-PEG-COOH PLGA-NHS
##STR00004##
CHCl.sub.3
[0149] CH.sub.2Cl.sub.2-PM=119.38 DIPEA N-Ethyldiisopropylamine
[(CH3)2CH]2NC2H5-PM=129.24 COOH-PEG-NH.sub.2
HCl.times.NH.sub.2-PEG-O--C.sub.3H.sub.6--COOH-PM PEG=3000da
Reagents
TABLE-US-00005 [0150] PLGA-NHS (reaction intermediate) 1 L
(synthesis) CHCl.sub.3 (Chloroform .gtoreq.99.5) 100 mL +
CHCl.sub.3 (Chloroform .gtoreq.99% stab. with 250 mL (washes) 0.75%
ethanol) 1.2 mL DIPEA (N,N-Diisopropylethylamine 99.5%) 7 g
COOH-PEG-NH.sub.2 (Polymer-hydrochloride form ratio) 1250 mL
Diethyl ether (.gtoreq.99.8%) 1250 mL Deionized water
Process
[0151] In a 2 L 3-necked flask equipped with mechanical stirrer,
under nitrogen flow, the resulting intermediate was dissolved in 1
L chloroform. 1.2 mL DIPEA were added to the system using a syringe
and subsequently 7 g COOH-PEG-NH.sub.2 (small additions). The
system was left under stirring under inert flow for about 90 h.
[0152] From the 3-necked flask, the yellow solution is transferred
into a 2L 1-necked flask and washed with 100 mL chloroform.
[0153] The product was concentrated to about 550 ml (distillate
volume CHCl3=650 ml) by means of a rotavapor. The product is
transferred from the 2 L flask to the 1 L 1-necked flask (washing
with 250 mL CHCl.sub.3).
[0154] The copolymer was precipitated and washed with 250 mL
(.times.5) of cold diethyl ether: at the beginning, the suspension
must be added slowly and shaken with a glass rod to prevent
over-saturation. At each wash, the system was rested in an ice bath
and then the supernatant was removed (opalescent suspension
containing quaternary salts and unreacted organic impurities).
[0155] The white solid of rubbery appearance was washed with 250 mL
(.times.5) deionized water to remove traces of unreacted
COOH-PEG-NH.sub.2.
[0156] The system was put under vacuum (liquid ring pump first and
oil pump thereafter), alternating vacuum drying (oil pump-trap at
-30.degree. C.), disintegration of the polymer to facilitate drying
and storage in a freezer overnight. The procedure is repeated until
no more weight loss is observed.
[0157] The product is stored in a freezer.
[0158] 86.80 g of polymer were recovered (yield of about 83%).
Example 7
[0159] Synthesis (PLGA-b-PEG-COOH 12-3 kDa) Step 1: Activation of
the Carboxylic Functionality with NHS
##STR00005##
Reagents
TABLE-US-00006 [0160] 7 g PLGA-COOH 7000-17000 (50:50 Poly
(DL-Lactide-co- glycolide), Carboxylate End Group) .fwdarw. 0.582
mmol 150 mL CH.sub.2Cl.sub.2 (Dichloromethane- >99.9%) for
solubilization of PLGA-COOH 0.27 g NHS (N-hydroxysuccinimide- 98%)
washed with 30 mL CH.sub.2Cl.sub.2 .fwdarw. 2.34 mmol 0.51 g DCC
(N,N Dicyclohexylcarbodiimide- 99%) washed with 50 mL
CH.sub.2Cl.sub.2 .fwdarw. 2.493 mmol 70 mL CH.sub.2Cl.sub.2
(Dichloromethane >99.9% to wash the 500 mL flask before
filtration on Buckner 550 mL Diethyl ether (Aldrich
.gtoreq.99.8%)
[0161] PLGA-COOH and 150 mL dichloromethane were added to a 500m
flask under nitrogen. After solubilization of the polymer, the NHS
was added (30 mL CH.sub.2Cl.sub.2 for funnel washing) and then DCC
was added--consecutive additions--50 mL CH.sub.2Cl.sub.2 for funnel
washing).
[0162] The system was left under stirring for about 24 hours in an
inert atmosphere.
[0163] The colorless solution (with white solid in suspension) was
filtered on Buckner into a 1 L tailed flask in order to remove
dicyclohexylurea. The 500 mL flask was washed with 70 ml
CH.sub.2Cl.sub.2.
[0164] The product was transferred to a pear-shaped 1-necked flask
and concentrated by a Buchi rotavapor, after about 1 h, a thick
white suspension was obtained.
Step 2: Conjugation of PLGA-NHS with NH2-PEG-COOH
##STR00006##
Reagents
TABLE-US-00007 [0165] PLGA-NHS (reaction intermediate) in thick
suspension 260 mL CHCl.sub.3 (Chloroform- Aldrich
.gtoreq.99.5%-Cat) for intermediate 20 mL CHCl.sub.3 for amino PEG
COOH washing 0.35 mL DIPEA (N,N-Diisopropylethylamine 99.5%) 1.82 g
COOH-PEG-NH.sub.2 (Polymer-hydrochloride form ratio) .fwdarw.
0.6066 mmol 520 mL Diethyl ether (.gtoreq.99.8%) 300 mL Deionized
water
Process
[0166] In a 500 mL flask under nitrogen, intermediate 100
(.times.2) and 60 mL CHCl3 were solubilized. 0.35 mL DIPEA were
added to the system using a syringe and subsequently 1.82 g
COOH-PEG-NH2 with 20 mL funnel washing CHCl3). The system was left
under stirring under inert flow for 96h.
[0167] From the 4-necked flask the suspension, filtered on Buckner
for the presence of brown and white residues, was transferred to a
500 mL 1-necked flask washing with a few mL chloroform.
[0168] The product is concentrated (Volume CHCl3 distillate=170 mL)
by means of a rotavapor. 120 mL cold diethyl ether (white
suspension-rubbing of glass rod-ice bath), 60 mL (white
suspension-ice bath), 80 mL (beginning of precipitation-ice bath),
60 mL (freezer for about 1 hour) are added to the yellow solution.
The product was washed with 100 mL (.times.2) cold diethyl ether.
At each wash, the system was rested in freezer and then the
supernatant was removed (opalescent suspension containing
quaternary salts and unreacted organic impurities). The three
fractions in ether were dried (1.20 g).
[0169] The white solid of rubbery appearance was washed with 100 mL
(.times.3) deionized water (ice bath) to remove traces of unreacted
COOH-PEG-NH2.
[0170] The copolymer (18.10 g) was placed under vacuum in Buchi
rotavapor and was then connected to the oil pump (ethanol-dry ice
trap) for about 4h (7.78 g--drying alternating with
disintegration). The product was subjected to disintegration and
then placed under vacuum, alternating stages of drying,
disintegration and freezer. The procedure is repeated until no more
weight loss is observed.
[0171] The product is stored in a freezer.
[0172] 7.52 g of polymer were recovered (yield of 88%-86%).
[0173] (2 g) P.M.:11300 g/mol-0.1769 mmol
[0174] (5 g) P.M.:15400 g/mol-0.3246 mmol
[0175] mmol PLGA COOH: 0.5015
[0176] g PLGA PEG COOH (mol.sub.2g*P.M.sub..2g)
(mol.sub.5g*P.M.sub..5g)=2.529+5.972=8.5 g
[0177] Calculations with PM=12000. Expected 8.74 g PLGA PEG
COOH
Example 8
Setup of the Nanobioreactor (NBR)
Reagents:
[0178] 40.0 g Fe3O4-DDA equal to 9.5e-04 mol Fe3O4; 220.0 mg
PLGA-b-PEG-COOH equal to 5e-06 mol polymer; 400 ml phosphate buffer
1 mM
[0179] After having solubilized 220.0 mg polymer in 5 mL THF, 40.0
g Fe3O4-DDA are injected in the organic solution. Using a 60 mL
syringe, the product is concentrated in a rotavapor to remove the
THF present. The process is stopped when there is no longer
formation and condensation of organic vapors.
[0180] The product is then concentrated and dialyzed with Cogent M
system with a Pellicon 2 mini 100 kDa membrane, according to the
following procedure:
[0181] Once the system has been drained, the suspension of NBR,
concentrated by a theoretical factor of 1.5 and then dialyzed with
2000 mL UP water buffered 10.sup.-3M is introduced.
[0182] It is further concentrated to a volume of 100 mL in Pellicon
XL system with a 500 kDa membrane after keeping the system in NaOCl
1:10 for 30 minutes.
[0183] The process is stopped once the theoretical concentration
factor of 20.times. has been reached (theoretical conc. of
inorganic: 1.0%).
[0184] It is then filtered with Millipore Sterivex 0.22 .mu.m
filters in PES. It is stored in refrigerator.
Characterization
DLS
TABLE-US-00008 [0185] % (volume Sample PDI Z-ave V-mean peak) notes
NBR 0.125 43.10 34.55 100 After (.+-.0.61) (.+-.1.30)
filtration
Zpotential
TABLE-US-00009 [0186] Sample Zpot % (Z peak) Notes NBR -43.2 100
After filtration
ICP
TABLE-US-00010 [0187] Sample Fe % % Fe.sub.3O.sub.4 mMolarity NBR
0.735 1.015 43.84
Stability in Culture Medium:
[0188] the sample is diluted to 1:20 (0.05% wt inorganic phase) in
DMEM+10% FBS+glutamine+antibiotic: it is clear without
aggregates.
DLS
TABLE-US-00011 [0189] Sample PDI Z-ave V-mean % (volume peak) NBR
(20X) 0.161 139.60 143.20 100 culture medium (.+-.1.06)
(.+-.3.06)
Example 9
Continuous Nanobioreactor Production (NBR)
Reagents:
[0190] 200 mg PLGA-b-PEG-COOH equal to 4.4e-06 mol polymer; 52.6 g
THF; 36.4 g Fe3O4-DDA equal to 8.6e-04 mol Fe3O4; 1000 mL H.sub.2O
MilliQ with phosphate buffer at pH 7.4=10.sup.-3M.
[0191] To a 100 mL Erlenmeyer flask, 0.2 g polymer PLGA-b-PEG-COOH
and 52.6 g THF are added, stirring until complete dissolution
(several minutes). Finally, 36.4 g Fe3O4-DDA are added.
Synthesis:
[0192] A double peristaltic pump system was set up after
calibration to perform the addition of the organic solution in
aqueous stream (THF/water volume ratio=1/10). The respective tubes
draw the solution directly from the lungs containing the THF
solution (with PLGA-b-PEG-COOH and particles) and the phosphate
buffer solution prepared in 2.5 L bottle. The product is first
stripped in a rotavapor to remove the THF and then brought to
dryness; once the volatile component has evaporated, the product is
recovered.
[0193] The product is then concentrated and dialyzed with Cogent M
system with Pellicon 2 mini 100 kDa membrane.
[0194] Emptying the system, 254.5 g product are recovered.
[0195] Theoretical concentration factor: 4.7.times.
[0196] Theoretical concentration: 0.078%
[0197] Time needed for concentration and dialysis: 20'.
[0198] The product is further concentrated in Pellicon XL with 500
kDa membrane. Time needed to final concentration: 1 h.
[0199] Recovered: 11.51 g net of the dead volume inside the
membrane about 1-2 mL.
[0200] Theor. Conc. (considering the V.sub.dead 1.5 mL): 14%
Characterization
DLS
TABLE-US-00012 [0201] % (volume Sample PDI Z-ave V-mean peak) notes
NBR 0.125 41.8 34.2 100 After concentration (+0.4) (+0.9) in
Pellicon XL; diluted 0.05% in buffer ** for stability tests in
serum at 0.05% (theoretical)
ICP
TABLE-US-00013 [0202] Sample Fe % % Fe.sub.3O.sub.4 mMolarity NBR
1.078 1.490 64.35
Example 10
[0203] Set Up of Targeted Nanobioreactor with moAb (NBR_hERG)
Reagents:
[0204] 40.38 mL NBR equal to 0.533 .mu.mol --COOH; 2.32 mL
Sulfo-NHS 0.23 mM solution equal to 0.533 .mu.mol; 190 .mu.L
EDAC*HCl 28 mM solution equal to 0.053 mmol; 2.64 mL 1.52 mg/mL
hERG solution equal to 4.0 mg hERG (2.7e-08 mol); 1500 mL aqueous
solution of Na2HPO.sub.3=10.sup.-3M
[0205] To a sterile 250 mL vessel, 40.38 mL NBR are added and then
0.19 mL EDAC (0.028 M) and 2.32 mL Sulfo-NHS are added. After 40'
(at rest), 2.64 mL of the hERG1 solution (1.52 mg/mL=4.0 mg) are
diluted with 15.52 mL phosphate buffer 1 mM and this is added to
42.89 mL activated NBR. (V.sub.final=61.05 mL;
Fe.sub.3O.sub.4=0.056%). It is left to rest overnight.
[0206] The system is set up for the dialysis of the product using
the Pellicon XL 500 kDa membrane in PES. The system is washed with
300 mL H.sub.2O MilliQ, 400 mL of 0.5% sodium hypochlorite are
flown and the system is left to sterilize for about 30 min. It is
then washed with 400 mL buffer 1 mM.
[0207] The product is then concentrated to a volume of 22 mL,
setting the pump speed to about 15 mL/min (P=0.27 bar). Also the
first dialysis permeate is analyzed via BCA.RTM. test. It is
dialyzed with 100 mL (4 volumes) of buffer 1 mM at a speed of 15
mL/min (P=0.27 bar) and concentrated to a volume of 4.7 mL.
[0208] Finally, the product is filtered with 0.22 .mu.M filters in
PES. It is stored in refrigerator. The product thus obtained
exhibits a good stability after dilution in culture medium at 0.05%
wt; there are no aggregates or solid forms in flocculation visible
to the naked eye.
Characterization
DLS
TABLE-US-00014 [0209] % (volume Sample PDI Z-ave V-mean peak) notes
NBR_hERG 0.157 66.14 50.79 100 End (.+-.0.30) (.+-.0.32)
product
Zpotential
TABLE-US-00015 [0210] Sample Zpot % (Z peak) Notes NBR_hERG -40.0
100 End product Sample Fe % % Fe.sub.3O.sub.4 mMolarity NBR_hERG
0.226 0.312 13.465
ICP
Stability:
DLS
TABLE-US-00016 [0211] % (volume Sample PDI Z-ave V-mean peak) notes
NBR_hERG 0.179 136.7 137.6 100 End (.+-.0.7) (.+-.8.4) product
BCA Test
[0212] For the execution of the BCA test, the concentration of
NBR_hERG is normalized with respect to that of the corresponding
antibody-free (NBR), which therefore serves as a white. Once the
staining has developed by the addition of the relevant reagent, the
samples are analyzed by UV-vis spectrophotometer, then the
absorbance values are interpolated on the calibration curve
previously prepared (using a BSA standard) and the equivalent moAb
concentrations is extrapolated. The net amount of antibody present
on the NBR_hERG is calculated by subtracting the values of moAb
found in the eluate and in the white (NBR) from that corresponding
to the sample of NBR_hERG. See equation below:
C.sub.moAb(NBR_moAb)net=C.sub.moAb(NBR_moAb)-C.sub.moAb(NBR)-C.sub.moAb(-
eluate)
[0213] The following are the experimental values measured:
[0214] mAb Eluate=45 .mu.g/mL
[0215] mAb NBR=435 .mu.g/mL
[0216] mAb NBR_hERG=863 .mu.g/mL
[0217] Actual mAb (mAb NBR_hERG-mAb NBR)=428 .mu.g/mL
[0218] Ratio mAb/Fe.sub.3O.sub.4=0.14
Example 11
[0219] Set Up of Targeted Nanobioreactor with Fluo-Dyes
(NBR_Fluo)
Reagent Technical Specifications:
TABLE-US-00017 [0220] NBR [Fe.sub.3O.sub.4] = 0.081% [PLGA-b-PEG-
COOH] = 0.061% * 1,4-diaminobutane MW = 88.15 g/mol d = 0.877 g/mL
Alexa Fluor .RTM. 750 MW = 1300 g/mol (750 nm) Phosphate buffer in
H.sub.2O UP C = 1M; pH 7.4
Reagents:
TABLE-US-00018 [0221] 30.00 mL NBR (0.40 .mu.mol PPGC43-3.1) 1 mg
Alexa Fluor 750 (in 770 .mu.L .fwdarw. [1 mM]) 1500 mL H.sub.2O
MilliQ with 10.sup.-3 M phosphate buffer at 7.4 = Sulfo-NHS MW =
217.13 g/mol EDAC.cndot.HCl MW = 191.7 g/mol
Preparation of Fluo-NH2 Solution
[0222] The fluorophore is solubilized with 770 .mu.L DMSO obtaining
a solution 1 nmol/.mu.L. In a 12-mL vial, 4950 .mu.L 1 mM phosphate
buffer are added and 50 .mu.L of the fluorophore Alexa Fluor 750
solution (50 nmol) are added. It is then placed under magnetic
stirring and then 100 .mu.L of solution 132 .mu.g/mL
1,4-diaminobutane are added (corresponding to 150 nmol=13.2 g). The
solution is left to react for 24h, in the dark and under nitrogen
flow. The NH2-terminal fluorophore thus obtained will be used for
the subsequent synthesis step without being purified.
Preparation of the Sulfo-NHS Solution (0.23 mM)
[0223] Weigh exactly 5.0 mg of Sulfo-NHS and solubilize in a 100 mL
flask using 1 mM phosphate buffer.
Preparation of the EDAC Solution (0.028 mM)
[0224] To a 4 mL vial, 2.7 mg EDAC and 0.5 mL 1 mM buffer are
added. Cap and shake to facilitate mixing. This solution must be
prepared immediately before the reaction.
Synthesis:
[0225] To a 100 mL vessel, 30.00 mL NBR DF are added and 0.14 mL
EDAC (0.028 M) and 1.72 mL Sulfo-NHS (0.23 mM) are added.
[0226] Total volume: 30.00 mL+0.14 mL+1.72 mL=31.86 mL
[0227] After 40' (at rest), 2.64 mL of the Alexa Fluor 750 solution
(10 nmol/mL) are added.
[0228] It is left to rest for 4 h.
[0229] A control DLS is performed (NBR_Fluo TQ).
Purification:
[0230] The system is set up for the dialysis of the product using a
Pellicon XL 500 kDa membrane in PES and a peristaltic pump
Masterflex L/S with easy-load II head. The system is then
sterilized fluxing sodium hypochlorite at 0.5% and leaving to react
for about 30 min. After washing with sterile MilliQ water and
setting up the system with 1 mM phosphate buffer (also sterile),
the product is concentrated to a volume of 10 mL (collect 23 ml of
permeate) setting the pump speed to about 12 mL/min (P=0.25 mbar).
A rate of the first permeate is retained for the UV-VIS analysis.
It is then dialyzed with 40 mL (4 volumes) of buffer 1 mM working
at a speed of 12 mL/min (P=0.25 mbar). At this point, it is
concentrated to a volume of 6 mL.
[0231] Recovered: 5.00 g
[0232] Theoretical synthesis concentration factor: 5.8.times.
[0233] Theoretical concentration factor compared to NBR:
5.times.
Filtration:
[0234] The product is filtered with 0.22 .mu.M Millex filters in
PES. For the purification of the entire product, one filter is
needed. It is stored in refrigerator.
[0235] NBR_27_Fluo_01 DF recovered=4.49 g
Characterization
DLS
[0236] R0366/2013; R0368/2013
TABLE-US-00019 Sample PDI Z-ave V-mean % notes NBR_Fluo 0.147 55.14
42.03 100 DF, dil. 1:10 (.+-.0.59) (.+-.1.59) in buffer 1 mM
Z Potential
[0237] R0368/2013
TABLE-US-00020 Sample Zpot Zwidth Cond % Z QF Notes NBR_Fluo -42.0
18.3 0.233 100 2.28 DF, dil. 1:10 in buffer 1 mM
ICP
[0238] R0368/2013
TABLE-US-00021 Sample Fe % % Fe.sub.3O.sub.4 mMolarity NBR_ Fluo
0.243 0.336 14.532
UV-Vis
[0239] R0368/2013
TABLE-US-00022 Sample Abs .epsilon. Conc. (nmol/L) % linked Eluate
NBR_Fluo 0.125575 242000 558 27.1
Example 12
[0240] Set Up of Targeted Nanobioreactor with Fluo-Dyes
(NBR_Fluo)
Reagent Technical Specifications:
TABLE-US-00023 [0241] NBR [Fe.sub.3O.sub.4] = 0.081% [PPGC43-3.1] =
0.061% * 1,4-diaminobutane MW = 88.15 g/mol d = 0.877 g/mL Alexa
Fluor 750 MW = 1300 g/mol (750 nm) Phosphate buffer C = 1M; pH 7.4
in H2O UP
Reagents:
TABLE-US-00024 [0242] 30.00 mL NBR (0.40 .mu.mol PPGC43-3.1) 50.00
nmol Cyanine 5-1,4-diaminobutane (in 5.0 mL .fwdarw. [10 .mu.M])
1500 mL H.sub.2O MilliQ with phosphate 10.sup.-3 M buffer at 7.4 =
Sulfo-NHS MW = 217.13 g/mol EDAC.cndot.HCl MW = 191.7 g/mol
Preparation of Fluo-NH2 Solution
[0243] To a 12 mL vial, 5 mL 1 mM phosphate buffer, 50 nmol (1
bottle) Cyanine 5, NHS-ester and then 150 nmol (13.2 g)
1,4-diaminobutane are added. The solution is left to react for 24
h, in the dark, under magnetic stirring and nitrogen flow. The
NH2-terminal fluorophore thus obtained will be used for the
subsequent synthesis step without being purified.
[0244] nitrogen flow. The NH2-terminal fluorophore thus obtained
will be used for the subsequent synthesis step without being
purified.
Preparation of the Sulfo-NHS Solution (0.23 mM)
[0245] Weigh exactly 5.0 mg of Sulfo-NHS and solubilize in a 100 mL
flask using 1 mM phosphate buffer.
Preparation of the EDAC Solution (0.028 mM)
[0246] To a 4 mL vial, 2.7 mg EDAC and 0.5 mL buffer 1 mM are
added. Cap and shake to facilitate mixing. This solution must be
prepared immediately before the reaction.
Synthesis:
[0247] To a 50 mL sterile vessel, 30.00 mL NBR are added and 0.14
mL EDAC (0.028 M) and 1.72 mL Sulfo-NHS (0.23 mM) are added.
[0248] Total volume: 30.00 mL+0.14 mL+1.72 mL=31.86 mL
[0249] After 40' (at rest), 2.64 mL of the Cyanin-5-NH2 solution
(10 nmol/mL) are added.
[0250] It is left to rest for 4 h.
Purification:
[0251] The system is set up for the dialysis of the product using
the Pellicon XL 500 kDa membrane in PES.
[0252] At this point, the product is concentrated to a volume of 10
mL (collect 23 mL permeate). Setting the pump speed to about 12
mL/min (P=0.25 mbar); t=4'
[0253] Retain a rate of the first permeate for the UV-VIS
analysis.
[0254] Dialyze with 40 mL (4 volumes) of buffer 1 mM. v 12 mL/min
(P=0.25 mbar); t=11'
[0255] At this point, it is concentrated to a volume of 6 mL;
t=5'.
[0256] Recovered: 4.29 g
[0257] Theoretical synthesis concentration factor: 5.5.times.
[0258] Theoretical concentration factor compared to NBR:
4.8.times.
Filtration:
[0259] The product is filtered with 0.22 .mu.M Millex filters in
PES. For the purification of the entire product, one filter is
needed. It is stored in refrigerator.
[0260] NBR_Fluo recovered=4.11 g
Example 13
Production of Targeted Nanobioreactor with moAb and Fluo-Dyes
(NBR_hERG-Fluo)
Reagent Technical Specifications:
TABLE-US-00025 [0261] NBR [Fe.sub.3O.sub.4] = 0.821% [PPGC43-3.1] =
0.616% 1,4-diaminobutane MW = 88.15 g/mol d = 0.877g/mL Cyanine
5-NHS ester (650 nm) Phosphate buffer in C = 1M; pH 7.4 H.sub.2O UP
Sulfo-NHS MW = 217.13 g/mol EDAC.cndot.HCl MW = 191.7 g/mol hERG1
MW = 15000 g/mol
[0262] Reagents:
TABLE-US-00026 4.79 mL NBR (0.64 .mu.mol PPGC43-3.1) 25 nmol
Cyanine 5-NHS ester (in 2.5 mL .fwdarw. [10 .mu.M]) 2.7 mg EDAC 5.0
mg Sulfo-NHS 4.8 mg hERG (3.2 mL .fwdarw. 1500 .mu.g/ml) 1500 mL
H.sub.2O MilliQ with [ ] = 10.sup.-3 M phosphate buffer at pH
7.4
[0263] Preparation of Fluo-NH2 Solution
[0264] To a 12 mL vial, 5 mL 1 mM phosphate buffer, 50 nmol (1
bottle) Cyanine 5 NHS-ester are added, place under magnetic
stirring and then add 100 .mu.L of 13.2 mg/100 mL solution of
1,4-diaminobutane (corresponding to 150 nmol=13.2 .mu.g). The
solution is left to react for 24h, in the dark and under nitrogen
flow. The NH2-terminal fluorophore thus obtained will be used for
the subsequent synthesis step without being purified.
[0265] Preparation of the Sulfo-NHS solution (0.23 mM)
[0266] Weigh exactly 5.0 mg of Sulfo-NHS and solubilize in a 100 mL
flask using 1 mM phosphate buffer.
[0267] Preparation of the EDAC solution (0.028 mM)
[0268] To a 4 mL vial, 2.7 mg EDAC and 0.5 mL buffer 1 mM are
added. Cap and shake to facilitate mixing. This solution must be
prepared immediately before the reaction.
Synthesis:
[0269] To a sterile 100 mL vessel, 7.95 mL buffer 1 mM and then
4.79 mL NBR are added. The mixture is stirred gently to mix and
then 2.29 mL EDAC (0.028 M) and 2.79 mL Sulfo-NHS (0.23 mM) are
added.
[0270] Total volume: 7.95 mL+4.79 mL+2.29 mL+2.79 mL=17.83 mL
[0271] After 40' (at rest), 2.5 mL of the Cyanin 5-NH5 ester
solution (10 nmol/mL) are added. 3.2 mL of the hERG1 solution (1.5
mg/mL=4.8 mg) are then diluted in 52.32 mL phosphate buffer 1 mM
and added to the suspension containing activated NBR and
fluorophore. It is left to rest overnight.
Purification:
[0272] The system is set up for the dialysis of the product using
the Pellicon XL 500 kDa membrane in PES already used for NBR_19.
Wash with 300 mL H.sub.2O MilliQ, then flux with 400 mL of 0.5%
sodium hypochlorite and leave in hypochlorite for about 30 min.
Wash the system with 400 mL buffer 1 mM.
[0273] At this point, the product is concentrated to a volume of 20
mL (collect 50 mL permeate).
[0274] Setting the pump speed to about 14 mL/min (P=0.2 mbar);
t=15'
[0275] Retain a rate of the first permeate for the BCA test.
[0276] Dialyze with 80 mL (4 volumes) of buffer 1 mM. v 14 mL/min
(P=0.2 mbar); t=16'
[0277] At this point, it is concentrated to a volume of 10 mL;
t=4'.
[0278] Recovered: 6.46 g
[0279] Theoretical dilution factor compared to NBR: 1.3.times.
Filtration:
[0280] The product is filtered with 0.22 .mu.M Millex filters in
PES. For the purification of the entire product, one filter is
needed. It is stored in refrigerator.
[0281] NBR_hERG-Fluo recovered=7.79 g
Characterization
DLS
TABLE-US-00027 [0282] Sample Dates PDI Z-ave V-mean % notes
NBR_hERG1- 15-feb 0.118 47.39 38.25 100 DF, dil 1:10 Fluo
(.+-.0.28) (.+-.0.89) in buff. 1 mM NBR_hERG1- 15-feb 0.176 155.4
171.5 100 DF, dil 1:10 Fluo DMEM (.+-.11.8) (.+-.19.4) in DMEM All
In
Z Potential
TABLE-US-00028 [0283] Sample Dates Zpot Zwidth Cond % Z QF Notes
NBR_19_hERG1_ Fluo_01 DF 15-feb -42.4 7.4 0.256 100 2.26 DF, dil.
1:10 in buffer 1 mM
ICP
TABLE-US-00029 [0284] Sample Fe % % Fe.sub.3O.sub.4 mMolarity
NBR_hERG1-Fluo 0.320 0.442 19.081
Example 14
[0285] Production of Nanobioreactor Loaded with Active Ingredient
(NBR_PTX and NBR_hERG_PTX)
Reagent Technical Specifications:
TABLE-US-00030 [0286] PPGC43-3.1 (batch 5-A) 50:50 Mw = 43000; PEG
Mw 3000 Fe3O4-DDA [Fe.sub.3O.sub.4] = 0.55% PTX Discovery Fine
Chemicals Phosph. buffer 1 mM pH = 7.4 THF d = 0.89
[0287] Reagents:
TABLE-US-00031 35.6 g Fe3O4-DDA (40.0 mL) (195.8 mg
Fe.sub.3O.sub.4) 490 mg/L (in water) 212.6 mg PPGC43-3.1 490 mg/L
(in water) 21.2 mg PTX 400 ml phosph. buffer 1 mM (actual: 440 mL)
60 mL syringe 25G needle
Preparation of the THF Solution [with PLGA-PEG (5.5 mg/g), PTX
(0.55 mg/g) and Fe.sub.3O.sub.4 (5.5 mg/g)]
[0288] 212.6 mg PPGC43-3.1 are solubilized in 4 mL (4 mL vial) THF
and 21.2 mg PTX in 2.12 mL THF (4 mL vial) and this is added to
35.6 g Fe3O4-DDA in a 100 mL flask
Synthesis:
[0289] The THF solution is stacked in 400 ml of phosphate buffer 1
mM using a 60 ml syringe with 25G needle.
[0290] NBR_PTX obtained: 455.8 g
Stripping
[0291] The product is treated in a rotavapor to remove the THF
present. To this end, it is moved to a 1000 mL flask and the
following conditions are set:
[0292] Bath T 40.degree.
[0293] Pressure: 154 mbar
[0294] Revolutions: 80 rpm
[0295] After 1 h, once the evaporation of the volatile component
has finished, the product is recovered and weighed.
[0296] NBR_PTX Rotavap recovered=418.22 g (37.58 g THF removed)
Dialysis and Concentration:
[0297] The product is concentrated and dialyzed with AMICON system
with a 50 kDa membrane, according to the following procedure:
[0298] 1) wash with 50 mL osmotized H.sub.2O to remove the
impurities in the membrane [0299] 2) system wash with solution with
50 mL phosphate buffer in H.sub.2O UP, 10.sup.-3M.
[0300] Once the system has been drained, NBR_PTX is added and
concentrated to about 100 mL
[0301] Thereafter, 4 washings are carried out with 150 mL buff. 1
mM. Finally, it is concentrated to 75 mL discarding 45 mL
eluate.
[0302] Emptying the system, 59.40 g of product (NBR_PTX) are
recovered.
[0303] Theoretical concentration factor=7.7.times.
Filtration:
[0304] The product is filtered with a Millipore Sterivex 0.22 .mu.M
filter in PES (cylindrical filters).
Characterization
DLS
TABLE-US-00032 [0305] Sample PDI Z-ave V-mean % notes NBR_PTX 0.174
53.55 41.66 100 DC, dil. 1:10 in (.+-.0.58) (.+-.0.55) buffer 1
mM
ICP
TABLE-US-00033 [0306] Sample Fe % % Fe.sub.3O.sub.4 mMolarity
NBR_PTX 0.347 0.480 20.716
Stability:
[0307] The concentrated sample diluted 1:8 in DMEM+10%
FBS+glutamine+antibiotic: is limpid without aggregates
DLS
[0308] R0211/2013
TABLE-US-00034 Sample Dates PDI Z-ave V-mean1 % notes NBR_PTX DMEM
08-may 0.155 126.1 121.8 100 DC, dil. 1:8 in AI (.+-.2.2) (.+-.4.6)
DMEM All In
PTX Analysis
TABLE-US-00035 [0309] PTX:PLGA-PEG PTX Sample ratio FWR % LC % LE %
mg/mL NBR_PTX_10 1:10 5.5 2.1 40.0 142.57
Example 15
[0310] Production of Nanobioreactor Loaded with Active Ingredient
(NBR_PT-X and NBR_hERG_PTX)
Reagent Technical Specifications:
TABLE-US-00036 [0311] NBR_PTX [Fe.sub.3O.sub.4] = 0.48%
[PPGC43-3.1] = 0.36% * Phosphate buffer C = 1M; pH 7.4 in H.sub.2O
UP Sulfo-NHS MW = 217.13 g/mol EDAC MW = 155.24 g/mol d = 0.877
g/mL hERG MW = 150000 g/mol
Reagents:
TABLE-US-00037 [0312] 4.26 mL NBR_PTX_p10 DF (3.33*10.sup.-4
.mu.mol PPGC43-3.1) 5.0 mg Sulfo-NHS (in 100 mL .fwdarw. [0.23 mM])
20 .mu.L EDAC (in 4 mL .fwdarw. [28 mM]) 2.5 mg hERG (0.833 mL*3
mg/mL) 1500 mL phosphate buffer at pH 7.4 [ ] = 10.sup.-3M
Preparation of the Sulfo-NHS Solution (0.23 mM)
[0313] Weigh 5.0 mg of Sulfo-NHS and solubilize in a 100 mL flask
using 1 mM phosphate buffer.
Preparation of the EDAC Solution (0.028 mM)
[0314] To a 4 mL vial, 2.7 mg EDAC and 0.5 mL buffer 1 mM are
added. Cap and shake to facilitate mixing. This solution must be
prepared immediately before the reaction.
Synthesis:
[0315] To a sterile 40 mL vessel, 2.36 mL buffer 1 mM and then 4.26
mL NBR_PTX are added. The mixture is stirred gently to mix and then
1.19 mL EDAC (0.028 M) and 1.45 mL Sulfo-NHS are added.
[0316] Total volume: 2.36 mL+4.26 mL+1.19 mL+1.45 mL=9.26 mL
[0317] After 40' (at rest), the HERG1 solution obtained by diluting
0.833 mL of the hERG1 solution (3 mg/mL) with 27.25 mL buffer 1 mM
is added. (V.sub.final=37.34 mL; Fe.sub.3O.sub.4=0.056%).
[0318] It is left to rest overnight.
Purification:
[0319] The system is set up for the dialysis of the product using
the Pellicon XL 500 kDa membrane in PES. Wash with 300 mL H.sub.2O
MilliQ, then flux with 400 mL of 0.5% sodium hypochlorite and leave
in hypochlorite for about 30 min. Wash the system with 400 mL
buffer 1 mM.
[0320] At this point, the product is concentrated to a volume of 13
mL (collect 25 mL permeate).
[0321] Setting the pump speed to about 13 mL/min (P=0.2 mbar);
t=5'
[0322] Retain a rate of the first permeate for the BCA test.
[0323] Dialyze with 60 mL (4 volumes) of buffer 1 mM. v 13 mL/min
(P=0.2 mbar); t=14'
[0324] At this point, it is concentrated to a volume of 10 mL;
t=10'.
[0325] Recovered: 7.40 g
[0326] Theoretical synthesis concentration factor: 5.5.times.
[0327] Theoretical dilution factor Compared to NBR_PTX:
2.8.times.
Filtration:
[0328] The product is filtered with 0.22 .mu.m Sterivex filters in
PES. For the purification of the entire product, one filter is
needed. It is stored in refrigerator.
[0329] NBR_PTX recovered=6.88 g
Characterization
DLS
TABLE-US-00038 [0330] Sample Dates PDI Z-ave V-mean % notes NBR_PTX
26-oct 0.138 63.62 50.58 100 DF, diluted (.+-.0.58) (.+-.0.60) 1:10
in buff. 1 mM
Zpotential
TABLE-US-00039 [0331] Sample Dates Zpot Zwidth Cond % Z QF Notes
NBR_PTX 26- -37.3 13.8 0.231 100 2.17 DF, diluted oct 1:10 in buff.
1 mM
ICP
TABLE-US-00040 [0332] Sample Fe % % Fe.sub.3O.sub.4 mMolarity
NBR_PTX 0.191 0.264 11.400
[0333] Actual yield of the process=94.5%
Example 16
Incorporation of NBR in Lymphocytes
[0334] T cells and Jurkat cells are capable of incorporating the
nano particles with a method developed by the applicants. T
cells/Jurkat cells are loaded with NP at a concentration of 0.05%
in a suitable specific culture medium (medium). In order to form
the medium containing the NP, the NP are dispensed first and then
the specific culture medium. The medium is made up as follows:
[0335] RPMI 1640 MEDIUM, w 2.0 g/L NaHCO.sub.3--w/o L-Glutamine.
(BIOCHROM) (code: F1215)--500 mL added with: [0336] L-GLUTAMINE,
solution 200 mM (100.times.). (EUROCLONE) (code: ECB 3000D)--5.5 mL
without dilution [0337] SODIUM PYRUVATE, 100 mM (100.times.).
(Gibco) (code: 11360-039)--5.5 mL without dilution [0338] MEM NEAA
Minimum essential medium Non-Essential Aminoacids (100.times.).
(Gibco) (code: 11140-035)-5.5 mL without dilution [0339] GENTOMIL
(gentamicin) 80 mg/2 ml (AIC N.sup.o 029314059)--1 2 mL vial [0340]
2-MERCAPTOETHANOL. (Merck) (code: 444203)--use 5.5 mL
2-Mercaptoethanol as follows: 37 .mu.l 2-MERCAPTOETHANOL in 99.963
mL sterile H.sub.2O (final vol 100 mL) [0341] 10% fetal bovine
serum FBS: Fetal Bovine Serum, Qualified. (Sigma-Aldrich) (code:
F6178)
[0342] Where necessary, autologous serum of the patient or media in
the absence of serum will be used instead of fetal bovine
serum.
* * * * *