Patent Application
20200061214
The present disclosure provides compositions and methods for real-time oxygen measurement. More particularly, the present disclosure relates to oxygen-sensing compositions including a metalloporphyrin compound.
The circulatory system employs specialized oxygen-carrying molecules in the blood to deliver oxygen from the lungs to other tissues throughout the body. To function normally, every organ in the body must contain sufficient amounts of oxygen in every tissue. Therefore, differing oxygen levels in tissue can be indicative of tissue structure abnormalities, diseases, or defects, whether caused externally or genetically. Therefore, reliable and accurate measurement of the oxygen supply in mammal tissue is important to ensure that the supply of oxygen is adequate and the tissue is healthy. Existing products and standard of care methods to measure oxygenation can be invasive, poorly localized, subject to positioning difficulties and motion artifact, time-consuming, and expensive.
The use of fiber optic probes employing luminescence quenching for such biomedical measurements has become very popular because these probes are easy to insert, involve no electrical hazards, and are economical to produce. In a fiber optic probe employing luminescence quenching, light from a suitable source is transmitted through long, thin, optically conducting flexible fibers of glass, plastic, or other transparent material to a receptor terminal containing a luminescent dye on an oxygen permeable support medium. The light causes the dye to luminesce and oxygen present in the blood or tissue quenches the luminescence. The light is then returned along the optical fiber to a light measuring instrument containing photomultiplier or photodiode tubes and an electronic computing circuit for processing.
While a number of fiber-optic probe devices have been reported, none of these devices are entirely satisfactory. Many luminescent dyes are fluorescent dyes and require expensive instrumentation because such dyes have short emission lifetimes and are not highly sensitive to quenching. Most fluorescent dyes are also sensitive to several anesthetic gases, often present in patients requiring tissue oxygenation monitoring. Furthermore, the permeability and solubility of the physiological gas in the support medium for the dye is not always optimal for the particular instrumentation employed.
Accordingly, there exist a need for non-invasive, real-time, and continuous methods for monitoring oxygenation at any given location or anatomical site that would be challenging for existing products.
One aspect of the present invention includes an oxygen-sensing compound including a metalloporphyrin encapsulated within a polymer particle.
Another aspect of the disclosure provides an oxygen sensor composition including an oxygen-sensing compound embedded within a hydrogel carrier, wherein the oxygen-sensing compound includes a metalloporphyrin encapsulated within a polymer particle.
Another aspect of the disclosure provides an optical fiber device for the detection of oxygen in a deep body organ of a subject comprising: (i) an optic probe that is coated with an oxygen sensor composition of the disclosure; (ii) an optical fiber in electrical communication with the optic probe; and (iii) a remote detector in electrical communication with the fiber.
Another aspect of the disclosure provides a method for monitoring oxygenation (e.g., oxygen concentration and/or oxygen tension) in a subject, the method including:
Another aspect of the disclosure provides a method for monitoring oxygenation (e.g., oxygen concentration and/or oxygen tension) in a subject, the method including:
Another aspect of the disclosure provides a system for monitoring oxygenation (e.g., oxygen concentration and/or oxygen tension), the system comprising (i) an oxygen sensor composition of the disclosure; (ii) an excitation light source; and (iii) an instrument for measuring and reporting fluorescence or phosphorescence from the activated oxygen-sensing compound.
The accompanying drawings are included to provide a further understanding of the methods and compositions of the disclosure, and are incorporated in and constitute a part of this specification. The drawings illustrate one or more embodiment(s) of the disclosure, and together with the description serve to explain the principles and operation of the disclosure.
Before the disclosed processes and materials are described, it is to be understood that the aspects described herein are not limited to specific embodiments, apparati, or configurations, and as such can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and, unless specifically defined herein, is not intended to be limiting.
Throughout this specification, unless the context requires otherwise, the word “comprise” and “include” and variations (e.g., “comprises,” “comprising,” “includes,” “including”) will be understood to imply the inclusion of a stated component, feature, element, or step or group of components, features, elements or steps but not the exclusion of any other integer or step or group of integers or steps.
As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.
Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.
As used herein, the term “contacting” includes the physical contact of at least one substance to another substance.
As used herein, “treatment,” “therapy” and/or “therapy regimen” refer to the clinical intervention made in response to a disease, disorder or physiological condition manifested by a patient or to which a patient may be susceptible. The aim of treatment includes the alleviation or prevention of symptoms, slowing or stopping the progression or worsening of a disease, disorder, or condition and/or the remission of the disease, disorder or condition.
The term “effective amount” or “therapeutically effective amount” refers to an amount sufficient to effect beneficial or desirable biological and/or clinical results.
As used herein, the term “subject” and “patient” are used interchangeably herein and refer to both human and nonhuman animals. The term “nonhuman animals” of the disclosure includes all vertebrates, e.g., mammals and non-mammals, such as nonhuman primates, sheep, dog, cat, horse, cow, chickens, amphibians, reptiles, and the like. Preferably, the subject is a human patient.
In view of the present disclosure, the methods and compositions described herein can be configured by the person of ordinary skill in the art to meet the desired need. In general, the disclosed materials, methods, and apparati provide improvements in real-time, in vivo monitoring of oxygenation. The inventors found a very efficient measurement of oxygen tension and/or oxygen concentrations in various tissues of the subject using the oxygen sensor compositions of the disclosure.
For example, once the oxygen sensor is in place, oxygen tension and/or oxygen concentration can be measured non-invasively at the site of implantation. The sensors may be implanted in either the skin or subcutis via a single needle stick. The sensors allow for real-time monitoring of localized oxygen tension at the particular site where the sensors are implanted, and also allow for rapid detection of changes in oxygenation. In many clinical scenarios, such as surgical flaps, the maintenance of adequate skin oxygenation is crucial to the success of the surgery. Identification of hypoxia allows for timely corrective action to restore oxygenation and salvage a compromised flap. It is estimated that 6 to 25 percent of skin flaps require a secondary surgical intervention and around 10 percent of flaps fail. Non-invasive monitoring of changes in tissue oxygenation where the sensors are injected, thus, obviates the need for percutaneous and cabled monitoring. In addition, the oxygen sensor compositions of the disclosure may also be biodegradable.
Most optical oxygen sensors are based on the decrease in fluorescence or phosphorescence intensity of a fluorophore(s) (indicator(s)) when they are quenched by molecular oxygen in either gas phase or in dissolved form. Some sensors are based on the fluorescence or phosphorescence lifetime decrease upon exposure to oxygen. The relationship between the intensity or lifetime in the absence (I0, τ0) and presence (I, τ) of oxygen is described by the Stern-Volmer equations:
I
0
/I=1+KSV[O2] (1)
τ0/τ=1+ksv[O2] (2)
where Ksv is the Stern-Volmer quenching coefficient having a specific value for each fluorophore/quencher system. [O2] is the concentration of O2, and when in gas phase, it is the partial pressure of oxygen (pO2) and the oxygen solubility (concentration) in water (in ppm) while in aqueous phase. Because oxygen can quench the fluorescence of many fluorophores, the quenching of the fluorescence is used for the detection of oxygen.
A similar premise is used for the present disclosure. The fluorescence of the metalloporphyrin compounds of the present disclosure is also quenched through the binding of oxygen, and hence can be used for the detection of oxygen. The polymer modification of the metalloporphyrin enables the metalloporphyrin to become fluorescent. The spectral characteristics (e.g., excitable wavelengths and emittance wavelengths) of the oxygen-sensing compound are dependent on the type of porphyrin and/or transition metal and/or polymer used.
Thus, one aspect the present disclosure provides an oxygen-sensing compound including a metalloporphyrin encapsulated within a polymer particle. Such oxygen-sensing compounds enable the measurement of oxygen concentration in a non-invasive, real-time, and continuous manner.
The disclosure also provides an oxygen sensor composition including an oxygen-sensing compound embedded within a hydrogel carrier, wherein the oxygen-sensing compound includes a metalloporphyrin encapsulated within a polymer particle.
As provided above, the oxygen-sensing compounds incorporate metalloporphyrin compound that is capable of fluorescing and/or phosphorescing with an intensity and lifetime that correlates with the degree of oxygenation.
In certain embodiments, the metalloporphyrin may be a compound including a macrocyclic tetra pyrroles and their variations/modification that are able to incorporate a transition metal.
In some embodiments, the metalloporphyrin further comprises a transition metal. As used herein, the term “transition metal” refers to one of the 38 elements in groups 3 through 12 of the periodic table. Transition metals suitable for the compositions of the disclosure include, but are not limited to, candium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc, yttrium, zirconium, niobium, molybdenum, technetium, ruthenium, rhodium, palladium, silver, cadmium, hafnium, tantalum, tungsten, rhenium, osmium, iridium, platinum, gold, etc. In some embodiments, the transition metal is palladium.
In certain embodiments, the metalloporphyrin comprises palladium tetraphenyl-tetrabenzoporphyrin (PdTPTBP).
As provided above, the metalloporphyrin is encapsulated within a polymer particle in the oxygen-sensing compounds of the disclosure. The polymer may comprise any polymer capable of encapsulating the metalloporphyrin thereby providing stability and retaining (or helping to impart) desirable optical properties. Suitable polymers may include, but are not limited to, polyethylethylene, poly(butadiene), poly(β-benzyl-L-aspartate), poly(lactic acid), poly(propylene oxide), poly(ε-caprolactam), oligo-methacrylate, polystyrene, polycaprolactone, polylactide, polyglycolide, poly(ethylene oxide)-polyethylethylene, poly(ethylene oxide)-poly(butadiene), poly(ethylene oxide)-poly(ε-caprolactone), poly(ethylene oxide)-poly(lactic acid), poly(vinyl chloride), poly(methyl methacrylate), poly(propylene) combinations thereof, and the like. In certain embodiments, the polymer is selected from the group poly(vinyl chloride), poly(methyl methacrylate), poly(propylene), polystyrene, and combinations thereof. In certain embodiments, the polymer comprises polystyrene.
The person of ordinary skill in the art will appreciate that a given polymer may have a variety of molecular weights and structures. Unless otherwise indicated, a “molecular weight” as used throughout is “weight-average” molecular weight, MW. MW may be calculated by using the equation: ΣMi2ni/ΣMini, where ni is the number of molecules of molecular weight Mi. The MW can be determined using any known technique, such as light scattering, small angle neutron scattering, X-ray scattering, or sedimentation velocity. The structures provided herein represent a weight average structure over the sample of the polymer. The person of ordinary skill in the art will be able to distinguish between different polymers, as having substantially different average molecular weights, or substantially different structures.
In certain embodiments, the polymer has a MW of about 500 Da to about 20 kDa. For example, the polymer has a MW of about 1 kDa to about 10 kDa; or about 1 kDa to about 5 kDa, or about 5 kDa to about 10 kDa, or about 3 kDa to about 7 kDa.
In certain embodiments, metalloporphyrin may be loaded into the polymer particle in the range of about to about 0.5 wt % to about 30 wt %, based on the total weight of the polymer. For example, metalloporphyrin may be loaded into the polymer particle in the range of about 1 wt % to about 20 wt %, or about 1 wt % to about 18 wt %, or about 1 wt % to about 15 wt %, or about 1 wt % to about 12 wt %, or about 1 wt % to about 10 wt %, or about 1 wt % to about 8 wt %, or about 1 wt % to about 5 wt %, or about 5 wt % to about 20 wt %, or about 5 wt % to about 18 wt %, or about 5 wt % to about 15 wt %, or about 5 wt % to about 12 wt %, or about 5 wt % to about 10 wt %, or about 5 wt % to about 8 wt %, or about 10 wt % to about 20 wt %, or about 10 wt % to about 15 wt %, or about 15 wt % to about 20 wt %.
The polymer particle may be of various sizes. For example, in some embodiments, the particle is a microscale particle. For example, the particle has a diameter of more than 100 μm. In other embodiments, the particle is a microscale particle. For example, the particle has a diameter in the range of about 0.1 μm to about 100 μm; e.g., in the range of about 0.1 μm to about 50 μm, or about 0.1 μm to about 20 μm, or about 0.1 μm to about 10 μm, or about 0.1 μm to about 5 μm, or about 1 μm to about 100 μm, or about 1 μm to about 50 μm, or about 1 μm to about 20 μm, or about 1 μm to about 10 μm, or about 1 μm to about 5 μm, or about 1 μm to about 3 μm, or about 2 μm to about 3 μm, or about 10 μm to about 100 μm, or about 10 μm to about 50 μm. In yet another embodiment, the particle is nanoscale particle. For example, the particle has a diameter in the range of about 1 nm to about 100 nm; e.g., in the range of about 1 nm to about 50 nm, or about 1 nm to about 20 nm, or about 1 nm to about 10 nm, or about 1 nm to about 5 nm, or about 1 nm to about 3 nm, or about 2 nm to about 3 nm, or about 10 nm to about 100 nm, or about 10 nm to about 50 nm, or about 50 nm to about 100 nm.
The oxygen-sensing compound of the disclosure may be prepared by any method known in the art. In a non-limiting example, the metalloporphyrin (e.g., PdTPTBP) may be combined with a polymer (e.g., polystyrene) carrier in chloroform is then cast as a thin film which is allowed to dry. The resulting solid is a powder containing the oxygen-sensing compound (e.g., metalloporphyrin encapsulated in the polymer particle).
As provided above, the oxygen sensor composition of the disclosure includes a hydrogel carrier. Any suitable hydrogel can be used to encapsulate the particle. For medicinal uses, the hydrogel is preferably biocompatible. Further, the hydrogels may be capable of reversible deformation. For example, the hydrogel may be implanted dry (e.g., in a smaller size/amount) and allowed to swell once implanted. Importantly, the hydrogels may maintain their original 3-dimensional shape and size making them easier to implant. Further, the particles are not covalently bound to the hydrogel carrier.
The hydrogel carrier may comprise a second polymer. Examples of the second polymer include, but are not limited to, poly(ethylene glycol) (PEG), poly(ethylene glycol) diacrylate (PEGDA), poly(hydroxethyl methacrylate) (PHEMA), silicone, poly(dimethylsiloxane) (PDMS), alginate, agarose, hyaluronic acid/hyaluronan, and their various formulations. In some embodiments, the second polymer comprises PEG diacrylate (PEGDA) (and various forms thereof). In certain embodiments, PEGDA is in its native nanoporous form. In certain embodiments, PEGDA is in its microporous form.
In certain embodiments, the second polymer has a MW of about 500 Da to about 20 kDa. For example, the second polymer has a MW of about 1 kDa to about 10 kDa; or about 1 kDa to about 5 kDa, or about 5 kDa to about 20 kDa, or about 5 kDa to about 10 kDa, or about 3 kDa to about 10 kDa, or about 3 kDa to about 7 kDa.
The hydrogel carrier may a protein or peptide hydrogel, such as collagen, gelatin, fibrin, elastin, bovine serum albumin (BSA), human serum albumin (HSA), etc.
In some embodiments, the number of particles within the hydrogel carrier may be varied to account for numerous factors, such as amount of fluorescence or phosphorescence needed for detection (e.g., deep tissue), amount of O2 present in the sample, etc. In certain embodiments, the particle may be loaded into the hydrogel carrier in the range of about to about 1 wt % to about 50 wt %, based on the total weight of the hydrogel. For example, the particle may be loaded into the hydrogel carrier in the range of about to about 1 wt % to about 40 wt %, or about 1 wt % to about 30 wt %, or about 1 wt % to about 25 wt %, or about 10 wt % to about 50 wt %, or about 10 wt % to about 40 wt %, or about 10 wt % to about 25 wt %, or about 10 wt % to about 20 wt %, or about 25 wt % to about 50 wt %, or about 25 wt % to about 40 wt %, or about 25 wt % to about 30 wt %, or about 15 wt % to about 35 wt %, or about 20 wt % to about 30 wt %, or about 10 wt % to about 20 wt %, or about 10 wt % to about 15 wt %, or about 15 wt % to about 20 wt %.
The hydrogel of the disclosure may be prepared by any method known in the art. In a non-limiting example, hydrogels formed at room temperature may be made into nanoporous hydrogels. In another non-limiting example, hydrogels formed at below zero temperatures (e.g., −20° C.) may be made into microporous cryogels. The cold polymerization creates competition between polymerization and ice crystal formation which leads to its microporous structure, which increases the toughness of the hydrogel. This toughness allows for the hydrogel to be injected within a tissue of a subject.
In another non-limiting example, hydrogels formed in emulsions may be made into nanoporous microspheres. For example, the nanoporous microspheres may have a diameter in the range of about 10 μm to about 100 μm; e.g, in the range of or about 50 μm to about 100 μm, or about 10 μm to about 50 μm, or about 30 μm to about 80 μm. In this formulation, the polymer particles may also be incorporated into microspheres through the use of an oil/water emulsion polymerization. This may also be similarly achieved via spray polymerization as well as microfluidics. In certain embodiments, the nanoporous microspheres may be further incorporated into other materials, such as suture materials as propyl propelene and poly urethane, and materials used for surgical devices, such as intravascular lines and indwelling devices.
The structure of the hydrogel may be used to control the degradation time of the oxygen-sensing compounds. For example, in certain embodiments, the second polymer monomers may incorporate variable numbers of bonds cleavable under physiological conditions, such as ester bonds (such aslactic acid, glycolic acid, or their combinations to the polymer) and disulfide bonds. For example, in certain embodiments, the second polymer may incorporate peptide sequences into the polymer backbone that can be selectively cleaved at different rates by various proteases including, but not limited to, matrix metalloproteinases, plasmins, and cathepsins. Each of these modes of degradation can be done in conjunction with any of the polymerization schemes.
In certain embodiments, the sensor composition of the disclosure may further include one or more different types of sensing compounds such as, but not limited to those specific for pH, CO2, O2, potassium, sodium, lactate, creatinine, glucose, urea, etc.
It will further be appreciated by persons skilled in the art that the oxygen sensor compositions of the disclosure may also comprise a suitable pharmaceutical excipient diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice (for example, see Remington: The Science and Practice of Pharmacy, 19th edition, 1995, ed. Alfonso Gennaro, Mack Publishing Company, Pennsylvania, USA).
For example, for application topically, e.g. to the skin or a wound site, the compositions of the present disclosure may be formulated as a suitable ointment containing the active compound suspended or dissolved in, for example, a mixture with one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene polyoxpropylene compound, emulisifying wax and water. Alternatively, they can be formulated as a suitable lotion or cream, suspended or dissolved in, for example, a mixture of one or more of the following: mineral oil, sorbitan monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, e-lauryl sulphate, an alcohol (e.g. ethanol, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol) and water. In certain embodiments, the formulation (e.g. lotion, solution, cream, gel or ointment) is water-based.
The oxygen sensor compositions of the disclosure may also be formulated for parenteral administration (for example, for administration intravenously, intra-arterially, intraperitoneally, intrathecally, intraventricularly, intrasternally, intraeranially, intra-muscularly or subcutaneously (including via an array of fine needles or using needle-free Powderject® technology), or by infusion techniques). In some embodiments, the oxygen sensor compositions of the disclosure may be in form of a cryogel as described herein, where the oxygen sensor composition is suspended in a cryogel and injected into the subject. In other embodiments, the oxygen sensor compositions of the disclosure may take the form of a sterile aqueous solution which may contain other substances, for example, an enough salts or glucose to make the solution isotonic with blood. The aqueous solutions should be suitable buffered (preferably to a pH of from 3 to 9), if necessary. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well-known to those skilled in the art.
Another aspect of the disclosure provides an optical fiber device. The optical fiber devices of the disclosure may be placed in locations that would not normally be directly optically accessible, such as in, but not limited to, deep body organs like the heart, lungs, liver, and kidneys. Referring to
The optical fiber 2 may be an optical fiber bundle. The bundle may include optical fibers covered by protective layers. Generally, the optical fiber bundle may include several thousands to several hundred and thousand optical fibers which are several μm in diameter, respectively. Both ends of the optical fiber 2 may be equipped with reflection-proofed glass plates to prevent reflection at both ends.
It is also within the scope of present disclosure that the oxygen sensor composition of the disclosure described herein may also be incorporated into/onto other medical devices where the real-time monitoring of oxygen concentration would be desirable. Such device may include, but are not limited to, endotracheal tubes (to monitor real-time lung oxygenation), arterial blood lines (to monitor real-time arterial blood gases), intravenous lines, central venous catheters, contact lenses, urinary catheters, surgical sutures, pacemakers, all forms of implantable devices, and orthopedic fixation devices. The oxygen sensor composition of the disclosure may also be incorporated into wound dressings and wound vacuum dressings thereby allowing for the real-time measurement of oxygenation in the wound. The oxygen-sensing compounds according to the present disclosure may also be used in numerous neurological applications, including, but not limited to, the continuous oxygen monitoring in cases of subarachnoid hemorrhages and intracranial hemorrhages. Further, the oxygen-sensing molecules (in, for instance, a hydrogel) can be implanted to predict/monitor peripheral vasospastic disease such as Raynaud's disease, acrocuanosis, livedo reticularis, and the like.
One aspect of the disclosure provides methods for monitoring oxygenation in a subject. In certain embodiments, such methods include: (i) administering to a subject a therapeutically effective amount of an oxygen sensor composition of the disclosure; (ii) activating an excitation light source to excite the oxygen-sensing compound; (iii) measuring the fluorescence or phosphorescence (e.g., the emitted light) from the oxygen-sensing compound; and (iv) calculating the concentration of oxygen from the measurement. In certain embodiments, such methods include: (i) coating a tip of an optic probe with an oxygen sensor composition of the disclosure; (ii) inserting the optic probe into the desired deep tissue of the subject; (iii) activating an excitation light source to excite the oxygen-sensing compound; (iv) measuring the fluorescence or phosphorescence (e.g., the emitted light) from the oxygen-sensing compound; and (v) calculating the concentration of oxygen from the measurement.
The methods of the disclosure allow for real-time and/or continuous measurement. The methods of the disclosure also allow for in vivo measurement in the tissue.
In certain embodiments, the measurement may be in the deep tissue.
In some embodiments, the deep tissue is selected from the group consisting of heart, lungs, liver, kidneys and combinations thereof.
The methods of the disclosure may also be used clinically in many diseases requiring monitoring of oxygen concentration in a certain organ or tissue. For example, in clinical scenarios like monitoring flap viability in the postoperative period, the oxygen sensor composition of the disclosure will immediately detect any vascular compromise through detecting decreases in oxygenation and thus can warrant an intervention to salvage the flap. Accordingly, in certain embodiments, the tissue is a surgical flap, replanted tissue, or transplanted organ of a subject.
Yet another application is in peripheral artery disease, where a plaque forms that can block arteries and require implantation of a vascular bypass. In such embodiments, the oxygen sensor compositions according to the disclosure are used to monitor oxygen concentration of affected tissues. Hence, another embodiment of disclosure provides methods wherein the tissue is in and around the peripheral arteries.
The oxygen sensor compositions of the disclosure may be included in numerous other methods that require the real-time monitoring of oxygen concentration. Such methods include, but are not limited to, free tissue transfer, organ transplants, hand and digital replant procedures, diabetic ulcers, pulmonary diseases (e.g., COPD), the monitoring of tumor response to treatment (and the prediction of prognosis and/or drug efficacy), skin grafts, vascular grafts and vascular bypass surgeries, dialysis shunts, the monitoring of decubitus ulcers, the monitoring of cyanosis in newborns, the monitoring if tongue necrosis during endotracheal intubation, and the like.
Further, it is within the scope of the present disclosure that the oxygen sensor compositions described herein may also be incorporated into non-in vivo/biological uses, such as for the monitoring of cell/bacterial cultures, monitor the viability of stem cells, incorporating into tattoo ink, and the like. The oxygen sensor compositions of the disclosure also have applicability in the monitoring of oxygen concentrations in open/closed systems, such as water flow, fermentation, bioreactors and the like.
Another aspect of the disclosure provides systems for monitoring oxygenation. Such systems include (i) an oxygen sensor composition of the disclosure; (ii) an excitation light source; and (iii) an instrument for measuring and reporting fluorescence or phosphorescence from the activated oxygen-sensing compound.
The systems of the disclosure allow for real-time and/or continuous measurement. The systems of the disclosure also allow for in vivo measurement in the tissue.
In certain embodiments, the systems may be adapted for measurement in the deep tissue. For example, the system of the disclosure may include the optic probe as described herein.
The composition and methods of the disclosure are illustrated further by the following examples, which are not to be construed as limiting the disclosure in scope or spirit to the specific procedures and compounds described in them.
The compositions disclosed herein can be made using procedures familiar to the person of ordinary skill in the art and as described herein. One of skill in the art can adapt the reaction sequences of the example below to fit the desired target molecule. Of course, in certain situations one of skill in the art will use different reagents to affect one or more of the individual steps or to use protected versions of certain of the substituents. Additionally, one skilled in the art would recognize that compositions of the disclosure can be synthesized using different routes altogether.
Poly(ethylene glycol) diacrylate (PEGDA) was synthesized by reacting 24 g poly(ethylene glycol) (PEG), 6000 MW (4 mmol, Fluka) with 1.27 mL acryloyl chloride (16 mmol, Sigma) and 1.12 mL triethyl amine (8 mmol, Sigma) overnight in 40 mL of dichloromethane (DCM) under argon atmosphere. The reaction was diluted with an additional 20 mL of DCM then washed with 2M K2CO3. The organic layer was collected and dried over MgSO4. The PEGDA could then be precipitated in diethyl ether, filtered and dried. NMR was used to evaluate the degree of acrylation.
As generally illustrated in
For nanoporous gels, the solution was mixed and pipette between two clean glass slides sandwiching a 1 mm thick Teflon mold and allowed to polymerize at room temperature for 30 minutes. The porphyrin-embedded hydrogels were then rinsed several times in milli-Q H2O and cut into the necessary dimensions for subsequent experiments. Hydrogels were stored at 4° C. in sterile water until use. Homogenous distribution of the metalloporphyrin was confirmed through fluorescent imaging via IVIS kinetic imaging system (
For microporous gels, the solution was pipette into a similar mold that had been pre-cooled at −20° C. and allowed to polymerize overnight at −20° C. The porphyrin-embedded hydrogels were then rinsed several times in milli-Q H2O and cut into the necessary dimensions for subsequent experiments. Hydrogels were stored at 4° C. in sterile water until use.
Spectroscopic Analyses.
Electronic absorption spectra were recorded on a Shimadzu UV-1700 spectrophotometer; sample was dissolved in THF solvent in a 10 mm quartz optical cell. Steady state emission spectra were recorded on a FLS920 spectrometer that utilized a xenon lamp (Xe900) as excitation light source and an extended red sensitive PMT (Hamamatsu R2658P side window photomultiplier, spectral range: 200-1010 nm) for detection. Emission spectra were corrected using calibration curve supplied with the instrument. Porphyrin compound was dissolved in THF solvent in 10 mm quartz optical cell, and investigated at ambient temperature in both deoxygenated and oxygenated condition. Deoxygenation was achieved through purging with dry argon gas over ˜30 min. The optical density at excitation wavelengths is ˜0.1 (
Nanosecond Spectroscopic Analysis.
The photoluminescence lifetime was acquired utilizing an Edinburgh Instruments LP920 Laser Flash Photolysis Spectrometer and Edinburgh L900 Software. Pump pulses were generated from a Q-switched Nd:YAG laser (Quantel, Brilliant) and a dual-crystal OPO (OPOTEK, Vibrant LDII). The temporal width of the pump pulses was ˜5 ns; the energy of the pulses exiting the OPO was controlled using neutral density filters. A Xe flash-lamp was used as a white light probe source. Both the LP920 and Opotek OPO are computer interfaced and controlled by the L900 software. Kinetics reported derive from data acquired over ˜20-50 scans. Samples were prepared in 1 mm quartz cells and purged with dry argon gas or air prior to excitation. Excited-state lifetimes were calculated via simple exponential fitting using Origin software. (
Combining the oxygen-sensitive porphyrins with polystyrene led to the formation of microparticles with spectroscopic properties which are different from the original oxygen-sensing porphyrin. These new resulting spectroscopic propertied allowed for enhancing the signal and obtained from the resulting compound, and allowed for a favorable shift in the absorption and emission wavelength, as well as increasing the lifetime of fluorescent decay (phosphorescence) which increases the usability for deeper implantation and usability of the sensors.
The resulting polystyrene-encapsulated porphyrins demonstrate more steady state fluorescence intensity as opposed to the porphyrin alone (
To determine the responsiveness and sensitivity, the non-implanted polystyrene-encapsulated porphyrin sensors were placed in a closed system that was sequentially purged with 100% CO2 followed by 100% O2. This process was then repeated and allowed to equilibrate to ambient oxygen. Here, the changes in ambient oxygen directly correlated to changes in the porphyrin's phosphorescence lifetime to generate an effective oxygen concentration (
In addition, these same sensors were then placed in normal saline solution for about 9 months. The signal and modulation from these molecules did not show unexpected variation or decreased signal.
Porphyrin-PEG-PQ was created by encapsulating the porphyrin/polystyrene microparticles in a PEGDA hydrogel that incorporates a matrix metalloproteinase (MMP)-sensitive peptide, GGGPQGIWGQGK (SEQ ID NO: 1; abbreviated PQ). Here, mono-acrylate-poly(ethylene glycol)-succinimidyl valerate was coupled to the amine-terminus of the peptide as well as to the C-terminus via the terminal lysine residue. The porphyrin/polystyrene microparticles (average mean particle size of 2.5 μm) were added to the generated PEG-PQ-PEG diacrylate macromer, and the mixture was then crosslinked in the presence of a chemical initiator (ammonium persulfate/tetramethylethylenediamine). This created a highly crosslinked hydrogel network that resists protein adsorption and permits the selective degradation of the hydrogel carrier.
It is expected that the hydrogel degrades within one month post implantation. The degraded poly(ethylene glycol) is cleared, leaving the <0.2 μl of porphyrin/polystyrene particles behind. This remaining particle mixture equates to about 10 μg of porphyrin (assuming a 5 μL hydrogel implant), which is about 1.2 μg of palladium coordinated within the porphyrin. This amount of palladium is much below the accepted levels for parenteral palladium administration (i.e., parenteral administration not exceeding 10 μg/day and oral administration not exceeding 100 μg/day). Therefore, the compositions of the disclosure permit pre-procedure baseline measurements, real-time tissue oxygen measurements during procedures, as well as post-procedural monitoring for up to several weeks. The biodegradable formulation is appropriate for short-term clinical applications, such as skin flaps, where oxygen tension monitoring is helpful during the first week post-procedure, after which neovascularization makes further monitoring unnecessary.
To determine the responsiveness and sensitivity of the sensors ex vivo, a fresh swine heart transplant model was used. Briefly, a fresh swine heart was harvested to be prepared for transplant in another pig. The sensors were implanted via a 19G needle in the heart that was being perfused on an ex vivo heart perfusion pump that keeps the heart pumping. The sensor was implanted in the myocardium of the left ventricle at a depth of ˜5 mm. The real-time readings fluorescent lifetime decay was obtained by a real-time fluorescent reader. Readings were obtained while the heart was being perfused and after perfusion was discontinued.
As shown in
To determine the responsiveness and sensitivity of the sensors, an ex vivo swine skin model was utilized. Briefly, a rectangular 10×10 cm rectangular piece of fresh swine back skin was obtained. The sensors of Example 2 (microporous cryogel) were implanted via a 19G-size needle intradermally (4 mm deep) and subcutaneously (6 mm deep) in the swine skin. Absolute fluorescence was imaged via the IVIS kinetic imaging system, and the fluorescent lifetime decay reading was done via a real-time fluorescence reader. The swine skin specimen, containing the implanted sensors was placed in a closed system that was sequentially purged with 100% O2 followed by 100% CO2.
As provided in
To determine the responsiveness and sensitivity of the sensors in vivo, three sensors of Example 2 (microporous cryogel) were implanted in vivo in swine tongue (n=3). Briefly, a swine acute tongue necrosis model was utilized using a yorkshire pig. While anesthesized, the sensors were implanted in the tongue of the pig a depth of 5 mm using a 19G-size needle, the sensors were implanted in the middle portion of the tip, center, and base of the tongue, respectively. A variety of manipulations, including vascular occlusion through a tourniquet, release of the tourniquet and tongue massage, was performed on the tounge. This experiment measured fluorescence lifetime, which is a measure of the time a fluorophore spends in the excited state before returning to the ground state by emitting a photon. The emission is then collected by the optical analysis device and values were converted into tissue oxygen tension in mmHg.
The sensors have appropriately responded to applying the tourniquets through reporting acute significantly decreased levels of oxygen. Three sensors were implanted at a consistent depth of 5 mm in the lateral margin of the tongue. The sublingual artery was occluded through applying a tourniquet, which was released, and the tongue was subjected to manipulations. The sensors have responded appropriately to manipulation of circulation, and reported the oxygenation as expected. The sensors have immediately detected the application of the tourniquet occluding the sublingual artery, then they detected the release of the tourniquet and the lower oxygenation resulted from damaging the sublingual artery. After releasing the tourniquets, the oxygen levels started rising again, as reported by the sensors. As provided in
While anesthetized, the sensors of Example 2 (microporous cryogel) were implanted in a Yorkshire swine in normal epidermis of the chest, right forelimb and left hindlimb of the pig. Baseline oxygen readings were obtained using real-time fluorescent lifetime decay measurements. While obtaining readings, euthanasia of the pig was performed through injecting intravenous euthanasia agent (KCl).
As provided in
To mimic a clinical scenario in a rodent model, the sensors were implanted in a rat model of random flap. A previously validated and published McFarlane rodent random flap model was used (Briggs, P. C., 1987. THE McFARLANE FLAP. Plastic and Reconstructive Surgery 80, 472.). Briefly, male Sprague-Dawley rats had the skin flap site outlined and three sensors were intradermally implanted at tip, middle and base of the impending flap as provided in
The sensors were able to predict flap necrosis on day 0 immediately after elevation of the flap as provided in
To assess how fast can the sensor respond to acute changes in oxygenation, a sensor was implanted in a rat myocutaneous flap model. Briefly, the sensors were implanted intradermally in the impending flap site. Superficial inferior epigastric artery (SIEA) myocutaneous flaps were surgically elevated. The SI EA flap was first outlined on the shaved skin of the right ventral abdomen by placing a 3×5 cm square template based on the location of the superficial inferior epigastric vessels. These vessels were carefully dissected to create a 3×5 cm island flap containing skin, subcutaneous fat, and panniculus carnosus muscle. Real-time fluorescent decay readings were obtained from implanted sensors of Example 2 (microprous cryogel), both at baseline and during vascular clamping of the feeding blood vessels.
Clinical observation of the flaps did not show any significant change in color and temperature of the flaps during or immediately after clamping of the feeding blood vessels. As shown in
It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be incorporated within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated herein by reference for all purposes.
This application claims the benefit of U.S. Provisional Patent Application No. 62/462,969, filed Feb. 24, 2017, all of which is incorporated herein by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2018/019536 | 2/23/2018 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62462969 | Feb 2017 | US |
