Cross-Reference to Related Applications

The present application claims priority to and the benefit of U.S. Provisional Application Serial Number 63/402,579, filed on August 31, 2022, the content of which is incorporated herein by reference in its entirety.

Field of the Invention

The invention relates generally to devices and methods for ultrasonic delivery of an agent to an internal tissue.

Background

The most common route of drug delivery is oral administration. Many drugs can be readily absorbed in the gastrointestinal (GI) tract, so oral administration allows them to enter the blood quickly and circulate systemically. In addition, oral administration is convenient and minimally invasive. Nonetheless, oral administration is not suitable for all drugs. For some drugs, the acidic conditions and harsh digestive enzymes of the GI tract degrade or inactivate the active pharmaceutical ingredient (API) before it can reach its target tissue. Other therapeutic agents, such as biological therapeutics ("biologies"), which generally consist of large macromolecules, are poorly absorbed in the GI tract. Absorption may also be limited if the patient has a diarrhea, which minimizes the duration of transit of the drug through the GI tract.

Ingestible ultrasonic drug delivery devices or capsules have been developed to overcome the difficulty of delivering certain drugs via the GI tract. Such devices incorporate the use of an ultrasound transducer, a reservoir that stores the drug, and a power source, such as a battery and drive circuitry, that drives the transducer. However, the utility of these fully self-contained devices is limited by a different set of technical obstacles. For example, the device must be small enough that it can be easily swallowed, yet large enough to accommodate the drug, transducer, drive circuitry, and battery. Conventional capsule electronics are highly volume inefficient, generally requiring multiple chips, packaging, and wires. The physical dimensions and mechanical characteristics of the device also dictate its biocompatibility with the gastrointestinal tract. For instance, the maximum size of a capsule’s rigid outer body is limited to the diameter of the smallest passage within the gastrointestinal tract. In addition, silver-oxide button batteries occupy significant real estate volume within ingestible capsules and often become the deciding factor for the device size. These factors constrain the quantity of drug that can be delivered by all-in-one ingestible ultrasonic drug delivery devices. Another consideration is that the battery can severely damage internal tissue if it were to make electrical contact with the tissue. The alkaline solution within ingested silveroxide button batteries can cause severe tissue damage in the mouth, vocal cord, trachea, or esophagus. Therefore, the device must contain material to electrically insulate the battery, which further restricts the drug-loading capacity of the device. Consequently, these factors largely limit the therapeutic potential of drug delivery via ingestible ultrasonic devices.

Summary

Aspects of the present disclosure may include a low power ingestible capsule drug delivery system for targeted or localized ultrasound-mediated drug delivery within the GI tract. The low power high-volume efficiency ingestible capsule drug delivery system may comprise one or more energy storage device, piezoelectric transducer, ultrasound transducer driver, drug carrier/reser- voir, at least one drug payload, and or at least one diagnostic unit. In various embodiments, the energy storage device may comprise a biocompatible battery or a biocompatible capacitor, a biocompatible supercapacitor, combinations thereof, or the like. In various embodiments, the ultrasound transducer driver may comprise one or integrated circuit (IC) or application-specific integrated circuit (ASIC) further comprising one or more internal resistor, transistor, bipolar transistor, field effect transistor (FET), capacitor, inductor, a finite state machine (FSM), and FPGA. In various embodiments, the said IC may function in conjunction with an external inductor or capacitor. In various embodiments, a crystal clock provides timing for the IC and an FSM for function control. In various embodiments, the said IC may comprise one or more drivers further comprising at least one charge-discharging unit. In various embodiments, the said IC may comprise one or more drivers further comprising at least one charge pump or switched-capacitor DC -DC boost converter unit. In various embodiments, the said IC may comprise one or more drivers further comprising at least one half-bridge transducer driver unit. In various embodiments, the battery may be cast into the rear part of the backing of said piezoelectric transducer which may be used to form the shape of a generated ultrasound emission. The electronics is integrated in one single ASIC which is mounted directly on a piezoelectric transducer. In various embodiments, the low power high-volume efficiency capsule may contain at least one piezoelectric transducer configured to operate in one or more modes, including but not limited to, thickness vibration, radial vibration, transverse vibration, combinations thereof, or the like. The ingestible capsule can perform one or more low power operations with high-volume efficiency incorporating a minimum number of electrical components for generating an ultrasound motive force, ultrasound field gradient, sonophoretic force, acoustic streaming, or cavitation within the GI tract using low frequency ultrasound.

Aspects of the present disclosure can include a method for generating an ultrasound motive force, ultrasound field gradient, sonophoretic force, acoustic streaming, or cavitation using a low power high-volume efficiency ingestible capsule drug delivery system for targeted or localized ultrasound-mediated drug delivery within the GI tract. In various embodiments, the method may comprise the use of one or more techniques for device operation using one or more biocompatible compact energy storage device, including but not limited to, a battery, a capacitor, a supercapacitor, combinations thereof, or the like. In various embodiments, the method may comprise the adiabatic charging of a piezoelectric transducer from a low voltage to a high voltage, preferably using an on-chip charge pump, boost converter, or inductive pump for high-volume efficiency and reduction of energy consumption. In various embodiments, the method may comprise the use of a half-bridge transducer driver. In various embodiments, the method comprises the use of one or more wave excitation, including but not limited to, a square wave, using one or more IC with a minimum number of components for high-volume efficiency and low power consumption. In various embodiments, the method may comprise the use of the piezoelectric transducer itself as storage capacitor. In various embodiments, the said transducer is slowly charged by a high voltage generation block, whereafter it is rapidly discharged to create one or more intermittent or continuous emission of ultrasound, preferably low frequency ultrasound to generate cavitation in an oral cavity or GI tract. In various embodiments, the method comprises the use of one or single edge excitation to reduce the complexity of the electronics of the low power and high-volume efficient ingestible capsule.

Aspects of the present disclosure may include a low power ingestible capsule drug delivery system and methods for targeted or localized ultrasound-mediated drug delivery within the GI tract. The ingestible capsule drug delivery system may comprise a diagnostic unit containing a sensor suite to determine the location of the capsule during transit through the GI tract. In various embodiments, the sensor suite comprises one or more physical, chemical, or bio-responsive sensor, including but not limited to, a pH, temperature, pressure, gas (e g., O2) chemical, biochemical, immuno-reactive, ultrasound, electromagnetic, magnetic field, CCD array, electrochemical, gravimetric, accelerometer, combinations thereof, or the like. In various embodiments, the methods for targeted drug delivery comprises calculating the time of transit to activate the said piezoelectric transducer to produce cavitation using low frequency ultrasound for drug delivery to at a specific location of the GI. In various embodiments, the methods for targeted drug delivery comprises the use of an internal clock of said IC to activate the said piezoelectric transducer to produce, for example, cavitation using low frequency ultrasound for drug delivery to at a specific location of the GI. In various embodiments, the methods for targeted drug delivery may comprise the calculation of a transit time to activate the said piezoelectric transducer to produce cavitation using low frequency ultrasound for drug delivery to at a specific location of the GI. In various embodiments, the methods for targeted drug delivery may comprise the determination of a pH, a pH change, a pH gradient to activate the said piezoelectric transducer to produce cavitation using low frequency ultrasound for drug delivery to at a specific location of the GI. In various embodiments, the methods for targeted drug delivery may comprise the determination of a body temperature, a body temperature change, a temperature gradient to activate the said piezoelectric transducer to produce cavitation using low frequency ultrasound for drug delivery to at a specific location of the GI. In various embodiments, the methods for targeted drug delivery may comprise determining a combination of a body temperature, pH, and or O2, temperature and pH and O2 change, or temperature, pH, or O2 gradient to activate the said piezoelectric transducer to produce, including but not limited to, cavitation using low frequency ultrasound for drug delivery to at a specific location of the GI. The said system and methods for targeted or localized ultrasound-mediated drug delivery within the GI tract preferably combine the functionalities of the diagnostic sensor suite and the capabilities to generate one or more adjustable controlled drug release profiles of one or more drug payload from said drug reservoir. In another aspect, the present disclosure provides methods of administering a therapeutic agent to a GI tissue of a subject by transporting the ingestible capsule to at least one specific location of the GI and the payload containing an encapsulated or non-encapsu- lated therapeutic agent that is activated by an ultrasound transducer within the capsule for control- released, pulsatile, non-pulsatile, intermittent, digital, or continuous local or targeted delivery of said agent from the payload or reservoir into GI tissue of the subject. Tn another aspect, said drug reservoir is configured to releasably retain at least one encapsulated therapeutic agent. In various embodiments, the ultrasound transducer may be positioned to transduce ultrasound waves in a particular direction relative to the reservoir of the ingestible capsule. The ultrasound transducer may be positioned to transduce ultrasound waves toward the reservoir. The ultrasound transducer is positioned to transduce ultrasound waves away from the reservoir. The ultrasound transducer is positioned to produce omnidirectional ultrasound waves through the reservoir. The reservoir is configured to releasably retain a liquid comprising a therapeutic agent or encapsulated therapeutic agent.

The ultrasound transducer of the ingestible capsule may produce an ultrasound signal with a defined frequency or within a defined frequency range. The ultrasound transducer may produce an ultrasound signal of from about 10 kHz to about 10 MHz, from about 10 kHz to about 1 MHz, from about 10 kHz to about 100 kHz, from about 20 kHz to about 80 kHz, from about 20 kHz to about 60 kHz, or from about 30 kHz to about 50 kHz. The ultrasound transducer may produce an ultrasound signal of less than 100 kHz, less than 80 kHz, less than 60 kHz, or less than 50 kHz. The ultrasound transducer may produce an ultrasound signal of about 20 kHz, about 25 kHz, about 30 kHz, about 35 kHz, about 40 kHz, about 45 kHz, about 50 kHz, about 55 kHz, or about 60 kHz. In various embodiments, the transducer comprises at least one, directional, planar, spherical, hemispherical, or omni-directional transducer.

The ingestible capsule may have a defined size or length. The ingestible capsule may have the longest dimension of less than about 3.0 cm, about 2.75 cm, about 2.5 cm, about 2.25 cm, about 2.0 cm, about 1.75 cm, or about 1.5 cm. The ingestible capsule may have a transverse dimension of less than about 1.2 cm, about 1.1 cm, about 1.0 cm, about 0.9 cm, or about 0.8 cm.

In an alternative embodiment, the ingestible capsule comprises a magnetic component. The magnetic component may comprise a ferromagnet or magnetic microparticles. In various embodiments, one or more permanent magnet may be positioned about a subject to attract the ingestible capsule to a specific location of the GI tract. In this aspect, the low power ingestible capsule drug delivery system further comprises a magnetic component to enable attraction to said external permanent to retain and secure the ingestible at a specific location within a GI tract.

In another aspect, the present disclosure provides methods of administering a therapeutic agent to a GI tissue of a subject by orally administering to a subject an ingestible capsule. In various embodiments, the therapeutic agent is encapsulated in at least one pH, thermal, electric, magnetic, electromagnetic wave, catalytic, piezo-catalytic, or ultrasound-responsive polymeric carrier, including but not limited to, microbubble, nanobubble, nanodroplet, nano emulsion, nanofiber, vesicle, micelle, or hydrogel sphere or coating. In various embodiments, the ingestible capsule may comprise one or more payload or reservoir containing at least one therapeutic agent encapsulated in at least one pH, thermal, electric, magnetic, electromagnetic wave, or ultrasound-responsive polymeric carrier. In various embodiments, the ingestible capsule may comprise one or more reservoir or payload containing at least one therapeutic agent encapsulated in at least one pH, thermal, electric, magnetic, electromagnetic wave, catalytic, piezo-catalytic, or ultrasound-responsive polymeric carrier. In various embodiments, the ingestible capsule may comprise a coating or scaffold on at least one internal or external surface, said coating or scaffold contains at least one therapeutic agent encapsulated in at least one pH, thermal, electric, magnetic, electromagnetic wave, catalytic, piezo-catalytic, or ultrasound-responsive polymeric carrier. In various embodiments, the ingestible capsule may contain an iron oxide particle-based biocompatible gel with a controlled architecture that can release its payload containing an encapsulated or non-encapsulated therapeutic agent when exposed to at least one AC magnetic field.

Brief Description of the Drawings

FIG. l is a pictorial of a low power ingestible capsule drug delivery system for targeted ultrasound-mediated drug delivery within the GI tract, according to an embodiment of the present disclosure.

FIG. 2 is a block diagram of a design of an IC for activating a piezoelectric transducer for low power battery operated ingestible capsule, according to an embodiment of the present disclosure.

FIG. 3 is a simplified schematic of an inductive boost converter, according to an embodiment of the present disclosure.

FIG. 4 is a simplified schematic of a discharge block of the low power battery operated ingestible capsule, according to an embodiment of the present disclosure.

FIG. 5 is a diagram of a miniaturized transducer driver comprising a charge pump and a half-bridge transducer driver, according to an embodiment of the present disclosure.

FIG. 6 is a block diagram of a method for targeted drug delivery using a low power ingestible capsule, according to an embodiment of the present disclosure. FIG 7 is a block diagram of a single-chip multi-sensor diagnostic unit, according to an embodiment of the present disclosure.

Detailed Description

It should be appreciated that all combinations of the concepts discussed in greater detail below (provided such concepts are not mutually inconsistent) are contemplated as being part of the inventive subject matter disclosed herein. It also should be appreciated that terminology explicitly employed herein that also may appear in any disclosure incorporated by reference should be accorded a meaning most consistent with the concepts disclosed herein.

It should be appreciated that various concepts introduced above and discussed in greater detail below may be implemented in any of numerous ways, as the disclosed concepts are not limited to any particular manner of implementation. Examples of specific implementations and applications are provided primarily for illustrative purposes. The present disclosure should in no way be limited to the exemplary implementation and techniques illustrated in the drawings and described below.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed by the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed by the invention, subject to any specifically excluded limit in a stated range. Where a stated range includes one or both endpoint limits, ranges excluding either or both of those included endpoints are also included in the scope of the invention.

As used herein, the term “includes” means includes but is not limited to, the term “including” means including but not limited to. The term “based on” means based at least in part on.

As used herein, the term “volume efficiency” means the amount of function per unit volume displaced by a capsule or device.

Ultrasound- Mediated Drug Delivery System and Components

Aspects of the present disclosure can include a low power ingestible capsule drug delivery system for targeted or localized ultrasound-mediated drug delivery within the GI tract. The high- volume efficiency low power ingestible capsule drug delivery system may comprise one or more low power energy storage device, piezoelectric transducer, ultrasound transducer driver, drug car- rier/reservoir, at least one drug payload, and optionally a diagnostic unit. The diagnostic unit further comprises a sensor suite to determine the location of the capsule during transit through the GI tract and for subsequent activation of the piezoelectric transducer at a targeted location. The in- gestible capsule can perform one more high efficiency low power operations with high-volume efficiency incorporating a minimum number of electrical components for generating an ultrasound motive force, ultrasound field gradient, sonophoretic force, acoustic streaming, or cavitation within the GI using low frequency ultrasound.

Referring now to FIG. 1, a pictorial 100 of a low power ingestible capsule drug delivery system for targeted ultrasound-mediated drug delivery within the GI tract is shown, according to various embodiments. In various embodiments, the high volume efficiency low power ingestible capsule drug delivery system 102 may comprise one or more energy storage device 104, piezoelectric transducer 106, ultrasound transducer driver 108, drug carrier/reservoir 110, at least one drug payload 112, and or at least one diagnostic unit 114. In various embodiments, the energy storage device may comprise a biocompatible battery or a biocompatible capacitor, a biocompatible supercapacitor, combinations thereof, or the like. In various embodiments, said biocompatible energy devices may incorporate one or more anode, cathode, and electrolytes to be biodegradable or safe for ingestion. In various embodiments, the anode-cathode-electrolyte combinations can be but not limited to: Mg/Fe/PCL/NaCl composite activated H2O, Mg/CuCl/SGF, Zn-Mg/Cu/SGF, Zn/SGF, AC-XMnCF/H O 1 MNa SCh, or Melanin/ XMnCh 1 MNa SCE. In various embodiments, energy storage device 104 may be a battery encapsulated for compatibility using, for example, Parylene deposited as a thin fdm. In various embodiments, the ultrasound transducer driver 108 may comprise one or integrated circuit (IC) 116 or ASIC further comprising one or more internal resistor, transistor, bipolar transistor, FET, capacitor, inductor, a finite state machine (FSM), or Field Programmable Gate Array (FPGA). In various embodiments, the said IC 116 may function in combination with an external inductor 118. In various embodiments, a crystal clock provides timing for the IC and an FSM for function control. In various embodiments, the said IC 116 may comprise one or more drivers further comprising at least one charge-discharging unit. In alternative embodiments, the battery 144 is cast into the rear part of the backing of ultrasound transducer 106 which may be used to form the shape of a generated ultrasound emission. The electronics is integrated in one single ASIC which may be mounted directly onto piezoelectric transducer 106. In various embodiments, the low power high-volume efficiency capsule 102 may contain at least one piezoelectric transducer 106 configured to operate in one or more modes, including but not limited to, thickness vibration, radial vibration, transverse vibration, combinations thereof, or the like. The ingestible capsule 102 can perform one or more high efficiency low power operations with high- volume efficiency incorporating a minimum number of electrical components for generating ultrasonic energy or intensity within the GI using low frequency ultrasound.

The transducer 106 delivers ultrasound energy at a frequency optimal for promoting entry of the therapeutic agent 112 into the tissue of the GI tract. The ultrasound transducer 106 may produce an ultrasound signal of from about 10 kHz to about 10 MHz, from about 10 kHz to about 1 MHz, from about 10 kHz to about 100 kHz, from about 20 kHz to about 80 kHz, from about 20 kHz to about 60 kHz, or from about 30 kHz to about 50 kHz. The ultrasound transducer 106 may produce an ultrasound signal of less than 100 kHz, less than 80 kHz, less than 60 kHz, or less than 50 kHz. The ultrasound transducer 106 may produce an ultrasound signal of about 20 kHz, about 25 kHz, about 30 kHz, about 35 kHz, about 40 kHz, about 45 kHz, about 50 kHz, about 55 kHz, or about 60 kHz. The design of the ingestible capsule 102 enables the transducer 106 to produce ultrasound energy at a desired power, frequency, or intensity.

In various embodiments, the ingestible capsule 102 may have a defined size or length. For example, and without limitation, the ingestible capsule 102 may have the longest dimension of less than about 3.0 cm, about 2.75 cm, about 2.5 cm, about 2.25 cm, about 2.0 cm, about 1.75 cm, or about 1.5 cm. The ingestible capsule 102 may have a transverse dimension of less than about 1.2 cm, about 1.1 cm, about 1.0 cm, about 0.9 cm, about 0.8 cm, about 0.7 cm, about 0.6 cm, or about 0.5 cm. The ingestible capsule 102 may have a radial dimension of less than about 1.2 cm, about 1.1 cm, about 1.0 cm, about 0.9 cm, about 0.8 cm, about 0.7 cm, about 0.6 cm, or about 0.5 cm.

Referring now to FIG. 2, a block diagram 200 of a design of an IC for activating a piezoelectric transducer for low power battery operation in an ingestible capsule is shown, according to various embodiments. In various embodiments, IC 202 comprises a control block 204, a boost converter 206, a discharge unit 208, and an oscillator 210. In various embodiments control block 204 controls the functionality of the IC 202 whereby parameters are set via digital inputs. In various embodiments, boost convert 206 is used to generate a high voltage on the piezoelectric transducer 212 prior to excitation with the use of an external or off-chip inductor 214. In various embodiments, discharge unit 208 activates the production of one or more ultrasound pulse or emission generated by one or more discharge of piezoelectric transducer 212 Tn various embodiments, the control of the functionality of IC 202 is handled by a finite state machine (FSM). The FSM relates all switching activities to its input clock signal derived from oscillator 210. In various embodiments, the core of the FSM can be, but not limited to a n-bit counter. In various embodiments, the output signals of the FSM controls one or more complete operational cycle of the chip. In various alternative embodiments, the control of the functionality of IC 202 is handled by an FPGA.

Aspects of the present disclosure may include an ultrasonic transducer driver that can overcome the dynamic range limitations of a low voltage battery supply by using an on-chip boost converter in combination with one external inductor to generate the required high voltage for excitation of the transducer. In various embodiments, the transducer itself is used as a storage capacitor, whereafter it is rapidly discharged to generate an ultrasound pulse. In various embodiments, the transducer is slowly charged by a high voltage generation block, whereafter it is rapidly discharged using one or more discharge controller to create an ultrasound pulse. This approach doesn’t require an external storage capacitor leading to a reduction in size, power consumption, number of transistors, and chip area.

Referring now to FIG. 3, a simplified schematic 300 of an inductive boost converter is shown, according to various embodiments. In various embodiments pumping is performed with a high voltage transistor M9302. During the pumping sequence transistor M7304 is on and transistor M8 306 is off. When the pumping cycle is complete, charge n 308 is pulled high so that M8 306 grounds the supply side of the inductor 310, equivalent to inductor 214 of FIG. 2, to prevent current built up in the inductor during the discharge of the piezoelectric transducer 312, equivalent to transducer 212 of FIG. 2, connected to the output of the pump. M7 304 and M8 308 are driven with unbalanced inverter chains to ensure that they are never carrying a short circuit current during switching. The transducer 312 is slowly charged by the high voltage generation block, whereafter it is rapidly discharged using one or more discharge block to create one or more ultrasound pulse.

Referring now to FIG. 4, a simplified schematic 400 of a discharge block is shown, according to various embodiments. In various embodiments, discharge unit 208 of FIG. 2 can be divided two or more equivalents block 402. In various embodiments, the division into blocks enables the adjustment of used discharge transistor size to the connected piezoelectric transducer 212 of FIG. 2. In various embodiments, the main component in the block can be a 12000/3 _m discharge transistor Ml 404. In various embodiments, Ml 404 can be a high voltage thin oxide n-channel transistor At a gate voltage of 3.6V, the transistor achieves a peak current of about 1 A. The gate capacitance presented by Ml is large, and care should be taken to use an appropriate transistor scaling to drive the gate. Care has also been taken during the layout process to ensure that the maximum allowed current densities in all layers of the chip are not exceeded during the discharge of the transducer. In various embodiments, a discharge is initiated by a high level on the pulse input, which propagates through the AND gate Al 406 to the buffers and to the discharge transistor Ml 404 which starts to discharge the node out. The task of the discharge control is to turn the discharge transistor Ml 404 off immediately when the out node has been discharged, to avoid holding the transducer clamped to ground level. A key component to achieve this is the controllable level shifter M2/M3 408 which transforms the high voltage on the out node to a level appropriate for the low voltage CMOS logic. The gate node of the inverter M4/M5 410 will hold a value of VDD - VGSM3 as long as the out node remains over approximately VDD - VGSM3 + VDS,sat M3. To avoid discharge of the out node during charge and hold, the level shifter 408 is turned on only when a discharge is initiated. When the out node has been discharged to a level approaching VDD - VGSM3, the gate node of M4/M5 410 starts to drop as it is pulled down by M2 when M3 turns off. The inverter M4/M5 410 switches and pulls the clock input of the D flip-flop high. This turns off the discharge transistor through the AND gate Al 406, as well as the sense circuit through AND gate A2 412. Before a new discharge cycle can be performed the D flip-flop has to be reset through reset.

Another aspect of the present disclosure is an ultrasonic transducer driver that can overcome the dynamic range limitations of a low voltage battery supply by using a DC-DC boost converter to generate the high voltage required by the system. The transducer driver is used to generate high-energy HV pulses with the control signals of a digital control block and the power supply of the DC-DC boost converter. Referring now to FIG. 5 a diagram 400a of a miniaturized transducer driver comprising a charge pump and a half-bridge transducer driver is shown, according to various embodiments. In various embodiments, a miniaturized transducer driver comprises a battery 402a, an ASIC 404a, an off-chip digital control block 406a, and a PZT transducer 408a. In various embodiments, battery 402a may comprise a lithium-ion battery with a nominal voltage, for example, approximately 4V. In various embodiments, in the ASIC 404a design, a half-bridge transducer driver 410a is used to drive the PZT transducer 408a, equivalent to transducer 106 of FIG. 1 to generate ultrasound. The output of the half-bridge transducer driver 410a equals the voltage that is applied across the PZT transducer 408a. Tn various embodiments, digital control signals for ASIC 402a are generated by the digital control block 406a of an FPGA. The control signals generated by the digital control block include a Clockl HS, Clockl LS for clock driver 412a and Clock2 HS, and Clock2 LS for clock driver 414a, said clock drivers driving charge pump 416a within the DC-DC boost converter 418a as well as the control signals LIUS HS for HV Level shifter 420a and LIUS LS for LS Gate Driver 422a the half-bridge transducer driver 410a. In various embodiments, transistor QI 424a is the high-side HS and transistor Q2 426a is the low side switch. In various embodiments, driver 410a may comprise of an internal bootstrap circuit, consisting of a diode DBOOT and a capacitor CBOOT, used to generate a bootstrap voltage VBOOT for HS Gate Driver 428a for the high-side operation of the half-bridge transducer driver 410a. The DC-DC boost converter 420a is used to generate the high voltage VPP needed by the transducer driver to interface with the PZT transducer 408a. In various embodiments, the output wave of the half-bridge transducer may be designed to operate at one or more frequencies depending on a desired output, including the drug dispensing, transport, or generation of ultrasound energy or intensity to produce, including but not limited to, cavitation or sonophoresis.

Aspects of the present disclosure may include methods for targeted or localized ultrasound- mediated drug delivery. The targeted delivery methods may comprise the use of a diagnostic unit having a sensor suite to determine the location of the capsule during transit through the GI tract. In various embodiments, the method may comprise the use of a sensor suite containing one or more physical, chemical, or bio-responsive sensor, including but not limited to, a pH, temperature, pressure, gas, chemical, immune-reactive, ultrasound, electromagnetic, magnetic field, CCD array, optical sensor, electrochemical, gravimetric, combinations thereof, or the like. In various embodiments, the methods for targeted drug delivery may comprise calculating the time of transit to activate the said piezoelectric transducer to produce ultrasound energy, intensity, or cavitation using low frequency ultrasound for drug delivery to at a specific location of the GI. In various embodiments, the methods for targeted drug delivery may comprise the use of an internal clock of said IC to activate the said piezoelectric transducer to produce ultrasound energy, intensity, or cavitation using low frequency ultrasound for drug delivery to at a specific location of the GI. In various embodiments, the methods for targeted drug delivery may comprise the calculation of a transit time to activate the said piezoelectric transducer to produce ultrasound energy, intensity, or cavitation using low frequency ultrasound for drug delivery to at a specific location of the GI. In various embodiments, the methods for targeted drug delivery may comprise the determination of a pH, a pH change, a pH gradient to activate the said piezoelectric transducer to produce ultrasound energy, intensity, or cavitation using low frequency ultrasound for drug delivery to at a specific location of the GI. In various embodiments, the methods for targeted drug delivery may comprise the determination of a body temperature, a body temperature change, a temperature gradient to activate the said piezoelectric transducer to produce ultrasound energy, intensity, or cavitation using low frequency ultrasound for drug delivery to at a specific location of the GI. In various embodiments, the methods for targeted drug delivery may comprise the determination of a combination of a body temperature and or pH, temperature and pH change, or temperature and pH gradient to activate the said piezoelectric transducer to produce ultrasound energy, intensity, or cavitation using low frequency ultrasound for drug delivery to at a specific location of the GI. The said system and methods for targeted or localized ultrasound-mediated drug delivery within the GI tract preferably combines the functionalities of the diagnostic sensor suite and the capabilities to generate one or more adjustable controlled drug release profiles of one or more drug payload from said drug reservoir. In another aspect, the present disclosure provides methods of administering a therapeutic agent to a gastrointestinal tissue of a subject by transporting the ingestible capsule to at least one specific location of the GI and the payload containing an encapsulated or non-encapsulated therapeutic agent that is activated by an ultrasound transducer within the capsule for control-released, pulsatile, non-pulsatile, intermittent, digital, or continuous local or targeted delivery of said agent from the payload or reservoir into gastrointestinal tissue of the subject.

Referring now to FIG. 6 a block diagram 500 of a method for targeted drug delivery using a low power ingestible capsule is shown, according to various embodiments. In one embodiment, the method comprises the use of a temperature and a pH sensor from said diagnostic sensor suite to determine a specific location with the GI for activating said ultrasound transducer (e.g., 106 of FIG. 1). In various embodiments, said temperature sensor can be a silicon temperature sensor. In various embodiments, said pH sensor can be a ISFET sensor requiring low power consumption. In various embodiments said chip may incorporated a sensor interface, a timer, and an FSM. In various embodiments, a patient swallows a capsule (Step 502), and the FSM is programmed for the capsule to analyze one or more GI landmarks, including but not limited to ingestion, pylorus, ileocecal valve, stomach, colon, or rectum. In various embodiments, ingestion is identified has having a rapid temperature rise and a rapid pH drop (> 3 pH values) (Step 504). In a preprogram FSM logic, a determination to deliver (Step 504a) a targeted amount of drug using transducer 106 of FIG. 1 to produce ultrasound energy, intensity, or cavitation to propel one or more released drug 112 of FIG. 1 into a targeted region of GI tissue. In various embodiments, the pylorus location is identified by a rapid pH rise (> 3 pH values (Step 506). In a preprogramed FSM logic, a determination is made to deliver (Step 506a) a targeted amount of drug using transducer 106 of FIG. 1 to produce ultrasound energy, intensity, or cavitation to propel one or more released drug 112 of FIG. 1 into a targeted region of GI tissue. In various embodiments, ileocecal valve is identified by a rapid drop to < 6.5 pH (Step 508). In a preprogrammed FSM logic, a determination to deliver (Step 508a) a targeted amount of drug using transducer 106 of FIG. 1 to produce ultrasound energy, intensity, or cavitation to propel one or more released drug 112 of FIG. 1 into a targeted region of GI tissue. In various embodiments, the sensor suit can be implemented in a CMOS process, including but not limited to, a 0.6 pm or 0.18 pm CMOS process.

Referring now to FIG. 7 a block diagram 600 of a single-chip multi-sensor diagnostic unit is shown, according to various embodiments. The single-chip multi-sensor diagnostic unit may comprise of a highly monolithic-integrated multimodal sensing system consisting of a capacitive based pressure sensor 602, a three-electrode electrochemical oxygen sensor 604, a solid-state based temperature sensor 606, their respective sensor interface circuits, a readout multiplexer 608, an analog-to-digital converter (ADC) 610, a digital controll er uni t 612, and a power management u nit 614. In v ari ou s emb odi m ent s , an interface circuit 616 for the capacitive pressure sensor includes a pseudo-differential sensor bridge, a two-stage capacitance-to-voltage converter (CVC) to convert the capacitance changes into voltage output, and a self-calibration circuit to auto-calculate the baseline for process variation compensation. In various embodiments, an oxygen sensor interface circuit has a negative feedback configuration operational amplifier as a potentiostat 618 and a transimpedance amplifier to convert the sensing current to output voltage. The solid-state temperature sensor has a bandgap reference and a proportional -to-absolute- temperature (PTAT) voltage generator 620 to sense the temperature variation and convert it into an output voltage. In various embodiments, multiplexer (MUX) 608 is used to feed the three sensing signals to the 10-bit successive approximation (SAR) ADC 610 to perform real-time data sampling and quantization in a time-multiplexed manner. The digital controller circuit of controller unit 612 provides the configuration and control for the rest circuits, performs digital filtering of the sensor signals. In various embodiments, the power management circuit consists of low drop- out (LDO) voltage regulators to provide a 1.8 V voltage to supply the sensors and integrated circuits. In various embodiments, the solid-state temperature sensor and integrated circuits may be fabricated using a 0.18-pm 1.8-V CMOS process. In various embodiments, the single-chip diagnostic unit may be used to perform one or more steps or methods described in FIG. 6 of the present disclosure using one or more said pressure, oxygen, or temperature sensor. The single-chip diagnostic unit may be a stand-alone chip or may be combined with one or more said ASIC of the ingestible capsule of the present disclosure to minimize components and chip areas of the electronics resulting in the fabrication of a high efficiency low power and high-volume efficiency ingestible capsule.

An object of the present disclosure is the encapsulation of therapeutic agents with a liquid, mixture, scaffold, or responsive polymer for incorporation into a reservoir of ingestible capsule. In various embodiments, the therapeutic agent is encapsulated in at least one pH, thermal, electric, magnetic, electromagnetic wave, catalytic, piezo-catalytic, or ultrasound-responsive polymeric carrier, including but not limited to, microbubble, nanobubble, nanodroplet, nano emulsion, nanofiber, vesicle, micelle, or hydrogel sphere or coating. In various embodiments, microbubble, nanobubble, nanodroplet, nano emulsion, nanofiber, vesicle, micelle, or hydrogel sphere or coating of the present disclosure may be produced from, but not limited to, poly(lactic acid), poly(allyla- mine hydrochloride), perfluorocarbon, polyvinyl alcohol, poly(lactic-co-glycolic acid, perfluoroc- tanol-poly(lactic acid). In various embodiments, pH or ultrasound-responsive polymer may comprise a scaffold, gel, or vesicle produce from, but not limited to, self-assembled from a polyethylene oxide)- block-poly[2-(diethylamino)ethyl methacrylate-stat-2-tetrahydrofuranyloxy) ethyl methacrylate] [PEO-b-P(DEA-stat- TMA)] block copolymers, polyethylene glycol) (PEG) crosslinked glycol chitosan (GC), Pluronic copolymers, poly(N ,N-diethyl acrylamide) (pNNDEA), or the like. In various embodiments, polymers for nucleic acid delivery includes, but not limited to, PS, poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), and polyplexes of cationic polymers, polyplexes of reporter gene DNA and polyethyleneimine (PEI), poly(l-lysine)/DNA (PLL/DNA), the like, or combination thereof.

Administering a therapeutic agent

Methods of administration

The invention provides methods of administering a therapeutic agent to gastrointestinal tissue of a subject using the systems and devices described above. The methods include delivering ultrasound energy to a liquid at a frequency that produces bubbles within the liquid and causes ultrasound energy, intensity, or cavitation of the bubbles. Gentle implosion of the bubbles produces shock waves that permeabilize cells and propel the agent from the liquid into the tissue. The use of ultrasound to cause cavitation to deliver agents to tissue is described in, for example, Schoellhammer, C. M., Schroeder, A., Maa, R., Lauwers, G. Y., Swiston, A., Zervas, M., et al. (2015). Ultrasound-mediated gastrointestinal drug delivery. Science Translational Medicine, 7(310), 310ral68-310ral68, doi: 10.1126/scitranslmed.aaa5937; Schoellhammer, C. M & Traverse, G., Low-frequency ultrasound for drug delivery in the gastrointestinal tract. Expert Opinion on Drug Delivery, 2016, doi: 10.1517/17425247.2016.1171841; Schoellhammer C. M., et al., Ultrasound-mediated delivery of RNA to colonic mucosa of live mice, Gastroenterology, 2017, doi: 10.1053/j.gastro.2017.01.002; and U.S. Publication Nos. 2014/0228715 and 2018/0055991, the contents of each of which are incorporated herein by reference.

In methods of the invention, the ultrasound signal may have a defined frequency. The ultrasound signal may have a frequency of from about 10 kHz to about 10 MHz, from about 10 kHz to about 1 MHz, from about 10 kHz to about 100 kHz, from about 20 kHz to about 80 kHz, from about 20 kHz to about 60 kHz, or from about 30 kHz to about 50 kHz. The ultrasound signal may have a frequency of less than 100 kHz, less than 80 kHz, less than 60 kHz, or less than 50 kHz. The ultrasound signal may have a frequency of about 20 kHz, about 25 kHz, about 30 kHz, about 35 kHz, about 40 kHz, about 45 kHz, about 50 kHz, about 55 kHz, or about 60 kHz.

In methods of the invention, the ultrasound signal may have a defined intensity. For example, and without limitation, the ultrasound signal may have an intensity of from about 0.001 W/cm ² to about 0.01 W/cm ² , from about 0.024 W/cm ² to about 0.04 W/cm ² , from about 0.014 W/cm ² to about 0.10 W/cm ² , from about 0.10 W/cm ² to about 0.5 W/cm ² , from about 0.5 W/cm ² to about .7500 W/cm ² , or from about 0.75 W/cm ² to about IW/cm ² .

In some embodiments, the ultrasound energy may be delivered as a pulse, i.e., it may be delivered over a brief, finite period to minimize damage to the agent being delivered by the ultrasound energy. For example, and without limitation, the pulse may be less than 20 minutes, less than 10 minutes, less than 5 minutes, or less than 10 minutes. For example, and without limitation, the pulse may be from about 10 seconds to about 3 minutes. The pulse may be about 10 minutes, about 5 minutes, about 3 minutes, about 3 minutes, about 1 minute, about 30 seconds, about 20 seconds, or about 10 seconds. The parameters of the ultrasound pulse, such as the frequency and/or duration, may be selected so that damage to the agent is limited to a certain fraction or percentage of the agent. For example, and without limitation, the ultrasound energy may result in breakdown of less than about 95% of the agent, less than about 90% of the agent, less than about 80% of the agent, less than about 70% of the agent, less than about 60% of the agent, less than about 50% of the agent, less than about 40% of the agent, less than about 25% of the agent, or less than about 10% of the agent.

The parameters of the ultrasound pulse, such as the frequency and/or duration, may be selected so that at least a minimum amount of the agent is transferred to the tissue. For example, and without limitation, the ultrasound energy may result in transfer of at least 1% of the agent, at least 2% of the agent, at least 5% of the agent, at least 10% of the agent, at least 20% of the agent, at least 30% of the agent, or at least 40% of the agent.

The methods may be used to deliver a therapeutic agent to a specific tissue in the GI tract. For example, the tissue may be buccal tissue, gingival tissue, labial tissue, esophageal tissue, gastric tissue, intestinal tissue, colorectal tissue, or anal tissue. The therapeutic agent may be targeted to a particular tissue in the GI tract. For example, the therapeutic agent may be targeted to the stomach, small intestine, large intestine (colon), rectum, or at a duct that enters the GI tract, such as a pancreatic duct or a common bile duct.

The methods may include administering an ingestible capsule to the subject. The ingestible capsule may be administered orally or rectally. The ingestible capsule may be administered via a duct that enters the GI tract.

The methods may include positioning the ingestible capsule within the subjects GI tract. For example, the ingestible capsule may be positioned in proximity to an affected region of the GI tract, such as an ulcer or inflamed region. The ingestible capsule may be positioned by applying a magnetic field to a portion of the subject’s GI tract from a device outside the subject’s body. The magnetic field may be applied using the transmitter. Alternatively, or additionally, the magnetic field may be applied from a magnetic device that is separate from the transmitter.

Therapeutic agents

The therapeutic agent may be any agent that provides a therapeutic benefit. For example and without limitation, suitable agents include alpha-hydroxy formulations, ace inhibiting agents, analgesics, anesthetic agents, anthelmintics, anti-arrhythmic agents, antithrombotic agents, antiallergic agents, anti-angiogenic agents, antibacterial agents, antibiotic agents, anticoagulant agents, anticancer agents, antidiabetic agents, anti-emetics, antifungal agents, antigens, antihypertension agents, antihypotensive agents, antiinflammatory agents, antimicotic agents, antimigraine agents, anti-obesity agents, antiparkinson agents, antirheumatic agents, antithrombins, antiviral agents, antidepressants, antiepileptics, antihistamines, antimuscarinic agents, antimycobacterial agents, antineoplastic agents, antithyroid agents, anxiolytics, asthma therapies, astringents, beta blocking agents, blood products and substitutes, bronchospamolytic agents, calcium antagonists, cardiovascular agents, cardiac glycosidic agents, carotenoids, cephalosporins, chronic bronchitis therapies, chronic obstructive pulmonary disease therapies, contraceptive agents, corticosteroids, cytostatic agents, cystic-fibrosis therapies, cardiac inotropic agents, contrast media, cough suppressants, diagnostic agents, diuretic agents, dopaminergics, elastase inhibitors, emphysema therapies, enkephalins, fibrinolytic agents, growth hormones, hemostatics, immunological agents, im- munosupressants, immunotherapeutic agents, insulins, interferons, lactation inhibiting agents, lipid-lowering agents, lymphokines, muscle relaxants, neurologic agents, NSAIDS, nutraceuticals, oncology therapies, organ-transplant rejection therapies, parasympathomimetics, parathyroid calcitonin and biphosphonates, prostacyclins, prostaglandins, psycho-pharmaceutical agents, protease inhibitors, magnetic resonance diagnostic imaging agents, radio-pharmaceuticals, reproductive control hormones, respiratory distress syndrome therapies, sedative agents, sex hormones, somatostatins, steroid hormonal agents, stimulants and anoretics, sympathomimetics, thyroid agents, vasodilating agents, vitamins, and xanthines. A more complex list of chemicals and drugs that can be used as agents in embodiments of the invention is provided in the Merck Index: An Encyclopedia of Chemicals, Drugs, and Biologicals Fifteenth Edition, Maryadele J O'Neil, ed., RSC Publishing, 2015, ISBN-13: 978-1849736701, ISBN-10 1849736707, the contents of which are incorporated herein by reference.

Therapeutic agents may be of any chemical form. For example, agents may be biological therapeutics, such as nucleic acids, proteins, peptides, polypeptides, antibodies, or other macromolecules. Nucleic acids include RNA, DNA, RNA/DNA hybrids, and nucleic acid derivatives that include non-naturally-occurring nucleotides, modified nucleotides, non-naturally-occurring chemical linkages, and the like. Examples of nucleic acid derivatives and modified nucleotides are described in, for example, International Publication WO 2018/118587, the contents of which are incorporated herein by reference. Nucleic acids may be polypeptide-encoding nucleic acids, such as mRNAs and cDNAs. Nucleic acids may interfere with gene expression. Examples of interfering RNAs (RNAi) include siRNAs and miRNAs. RNAi is known in the art and described in, for example, Kim and Rossi, Biotechniques. 2008 Apr; 44(5): 613-616, doi: 10.2144/000112792; and Wilson and Doudna, Molecular Mechanisms of RNA Interference, Annual Review of Biophysics 2013 42: 1, 217-239, the contents of each of which are incorporated herein by reference. Agents may be organic molecules of non-biological origin. Such drugs are often called small-molecule drugs because they typically have a molecular weight of less than 2000 Daltons, although they may be larger. Agents may be combinations or complexes of one or more biological macromolecules and/or one or more small molecules. For example, and without limitation, agents may be nucleic acid complexes, protein complexes, protein-nucleic acid complexes, and the like. Thus, the agent may exist in a multimeric or polymeric form, including homocomplexes and heterocomplexes.

An advantage of ultrasound-based delivery of therapeutic agents is the capacity to deliver large molecules, e.g., molecules having a molecular weight greater than 1000 Da. Thus, the therapeutic agent may have a minimum size. For example, and without limitation, the antigen may have a molecular weight of > 100 Da, > 200 Da, > 500 Da, > 1000 Da, > 2000 Da, > 5000 Da, > 10,000 Da, > 20,000 Da, > 50,000 Da, or > 100,000 Da.

The therapeutic agent may be provided in a liquid that promotes delivery of the therapeutic agent using the devices or systems provided herein. For example, the liquid may facilitate ultrasound-induced cavitation, iontophoresis, sonoporation, magneto-sonoporation, or electroporation. The liquid may be aqueous. The liquid may contain ions. The liquid may be an aqueous solution that contains one or more salts. The liquid may contain a buffer.

The therapeutic agent may be formulated. Formulations commonly used for delivery of biologic and small-molecule agents include drug crystals, gold particles, iron oxide particles, lipid- like particles, liposomes, micelles, microparticles, nanoparticles, polymeric particles, vesicles, viral capsids, viral particles, and complexes with other macromolecules that are not essential for the biological or biochemical function of the agent.

Alternatively, the therapeutic agent may be unformulated, i.e., it may be provided in a biologically active format that does not contain other molecules that interact with the agent solely to facilitate delivery of the agent. Thus, the agent may be provided in a non-encapsulated form or in a form that is not complexed with other molecules unrelated to the function of the agent. The agent may be a component of a gene editing system, such as a meganuclease, zinc finger nuclease (ZFN), a transcription activator-like effector-based nuclease (TALEN), or the clustered, regularly-interspersed palindromic repeat (CRISPR) system.

Meganucleases are endodeoxyribonucleases that recognize double-stranded DNA sequences of 12-40 base pairs. They can be engineered to bind to different recognition sequences to create customized nucleases that target particular sequences. Meganucleases exist in archaebacte- rial, bacteria, phages, fungi, algae, and plants, and meganucleases from any source may be used. Engineering meganucleases to recognize specific sequences is known in the art and described in, for example, Stoddard, Barry L. (2006) "Homing endonuclease structure and function" Quarterly Reviews of Biophysics 38 (1): 49-95 doi:10.1017/S0033583505004063, PMID 16336743; Grizot, S.; Epinat, J. C.; Thomas, S.; Duclert, A.; Rolland, S.; Paques, F.; Duchateau, P. (2009) "Generation of redesigned homing endonucleases comprising DNA-binding domains derived from two different scaffolds" Nucleic Acids Research 38 (6): 2006-18, doi: 10.1093/nar/gkpl l71. PMC 2847234, PMID 20026587; Epinat, Jean-Charles; Arnould, Sylvain; Chames, Patrick; Rochaix, Pascal; Desfontaines, Dominique; Puzin, Clemence; Patin, Amelie; Zanghellini, Alexandre; Paques, Frederic (2003-06-01) "A novel engineered meganuclease induces homologous recombination in yeast and mammalian cells" Nucleic Acids Research 31 (11): 2952-2962; and Seligman, L. M.; Chisholm, KM; Chevalier, BS; Chadsey, MS; Edwards, ST; Savage, JH; Veillet, AL (2002) "Mutations altering the cleavage specificity of a homing endonuclease" Nucleic Acids Research 30 (17): 3870-9, doi: 10.1093/nar/gkf495. PMC 137417, PMID 12202772, the contents of each of which are incorporated herein by reference.

ZFNs are artificial restriction enzymes that have a zinc finger DNA-binding domain fused to a DNA-cleavage domain. ZFNs can also be engineered to target specific DNA sequences. The design and use of ZFNs is known in the art and described in, for example, Carroll, D (2011) "Genome engineering with zinc-finger nucleases" Genetics Society of America 188 (4): 773-782, doi:10.1534/genetics.l 11.131433. PMC 3176093, PMID 21828278; Cathomen T, Joung JK (July 2008) "Zinc-finger nucleases: the next generation emerges" Mol. Ther. 16 (7): 1200-7, doi:10.1038/mt.2008.114, PMID 18545224; Miller, J. C ; Holmes, M. C ; Wang, J.; Guschin, D. Y.; Lee, Y. L.; Rupniewski, I.; Beausejour, C. M.; Waite, A. J.; Wang, N. S.; Kim, K. A.; Gregory, P. D.; Pabo, C. O.; Rebar, E. J. (2007) "An improved zinc-finger nuclease architecture for highly specific genome editing" Nature Biotechnology, 25 (7): 778-785, doi : 10.1038/nbtl 319, PMID 17603475, the contents of each of which are incorporated herein by reference.

TALENs are artificial restriction enzymes that have a TAL effector DNA-binding domain fused to a DNA cleavage domain. TALENs can also be engineered to target specific DNA sequences. The design and use of TALENs is known in the art and described in, for example, Boch J (February 2011) "TALEs of genome targeting" Nature Biotechnology 29 (2): 135-6, doi:10.1038/nbt.1767. PMID 21301438; Juillerat A, Pessereau C, Dubois G, Guyot V, Marechai A, Valton J, Daboussi F, Poirot L, Duclert A, Duchateau P (January 2015) "Optimized tuning of TALEN specificity using non-conventional RVDs" Scientific Reports, 5: 8150, doi:10.1038/srep08150. PMC 4311247, PMID 25632877; and Mahfouz MM, Li L, Shamimuz- zaman M, Wibowo A, Fang X, Zhu JK (February 2011) "De novo-engineered transcription activator-like effector (TALE) hybrid nuclease with novel DNA binding specificity creates doublestrand breaks" Proceedings of the National Academy of Sciences of the United States of America, 108 (6): 2623-8, Bibcode:2011PNAS, 108.2623M, doi: 10.1073/pnas.1019533108, PMC 3038751, PMID 21262818, the contents of each of which are incorporated herein by reference.

The CRISPR system is a prokaryotic immune system that provides acquired immunity against foreign genetic elements, such as plasmids and phages. CRISPR systems include one or more CRISPR-associated (Cas) proteins that cleave DNA at clustered, regularly-interspersed palindromic repeat (CRISPR) sequences. Cas proteins include helicase and exonuclease activities, and these activities may be on the same polypeptide or on separate polypeptides. Cas proteins are directed to CRISPR sequences by RNA molecules. A CRISPR RNA (crRNA) binds to a complementary sequence in the target DNA to be cleaved. A transactivating crRNA (tracrRNA) binds to both the Cas protein and the crRNA to draw the Cas protein to the target DNA sequence. Not all CRISPR systems require tracrRNA. In nature crRNA and tracrRNA occur on separate RNA molecules, but they also function when contained a single RNA molecule, called a single guide RNA or guide RNA (gRNA). The one or more RNAs and one or more polypeptides assemble inside the cell to form a ribonucleoprotein (RNP). CRISPR systems are described, for example, in van der Oost, et al., CRISPR-based adaptive and heritable immunity in prokaryotes, Trends in Biochemical Sciences, 34(8):401-407 (2014); Garrett, et al., Archaeal CRISPR-based immune systems: exchangeable functional modules, Trends in Microbiol. 19(11):549-556 (2011); Makarova, et al., Evolution and classification of the CRISPR-Cas systems, Nat. Rev. Microbiol. 9:467-477 (2011); and Sorek, et al., CRISPR -Mediated Adaptive Immune Systems in Bacteria and Archaea, Ann. Rev. Biochem. 82:237-266 (2013), the contents of each of which are incorporated herein by reference.

CRISPR-Cas systems have been placed in two classes. Class 1 systems use multiple Cas proteins to degrade nucleic acids, while class 2 systems use a single large Cas protein. Class 1 Cas proteins include CaslO, CaslOd, Cas3, Cas5, Cas8a, Cmr5, Csel, Cse2, Csfl, Csm2, Csxl 1, Csyl, Csy2, and Csy3. Class 2 Cas proteins include C2cl, C2c2, C2c3, Cas4, Cas9, Cpfl, and Csn2.

CRISPR-Cas systems are powerful tools because they allow gene editing of specific nucleic acid sequences using a common protein enzyme. By designing a guide RNA complementary to a target sequence, a Cas protein can be directed to cleave that target sequence. In addition, although naturally-occurring Cas proteins have endonuclease activity, Cas proteins have been engineered to perform other functions. For example, endonuclease-deactivated mutants of Cas9 (dCas9) have been created, and such mutants can be directed to bind to target DNA sequences without cleaving them. dCas9 proteins can then be further engineered to bind transcriptional activators or inhibitors. As a result, guide sequences can be used to recruit such CRISPR complexes to specific genes to turn on or off transcription. Thus, these systems are called CRISPR activators (CRISPRa) or CRISPR inhibitors (CRISPRi). CRISPR systems can also be used to introduce sequence-specific epigenetic modifications of DNA, such acetylation or methylation. The use of modified CRISPR systems for purposes other than cleavage of target DNA are described, for example, in Dominguez, et al., Beyond editing: repurposing CRISPR-Cas9 for precision genome regulation and interrogation, Nat. Rev. Cell Biol. 17(1): 5- 15 (2016), which is incorporated herein by reference.

The agent may be any component of a CRISPR system, such as those described above. For example and without limitation, the CRISPR component may be one or more of a helicase, endonuclease, transcriptional activator, transcriptional inhibitor, DNA modifier, gRNA, crRNA, or tra- crRNA. The CRISPR component contain a nucleic acid, such as RNA or DNA, a polypeptide, or a combination, such as a RNP. The CRISPR nucleic acid may encode a functional CRISPR component. For example, the nucleic acid may be a DNA or mRNA. The CRISPR nucleic acid may itself be a functional component, such as a gRNA, crRNA, or tracrRNA.

The agent may include an element that induces expression of the CRISPR component. For example, expression of the CRISPR component may be induced by an antibiotic, such as tetracycline, or other chemical. Inducible CRTSPR systems have been described, for example, in Rose, et al., Rapidly inducible Cas9 and DSB-ddPCR to probe editing kinetics, Nat. Methods, 14, pages 891-896 (2017); and Cao, et al., An easy and efficient inducible CRISPR/Cas9 platform with improved specificity for multiple gene targeting, Nucleic Acids Res. 14(19):el49 (2016), the contents of which are incorporated herein by reference. The inducible element may be part of the CRISPR component, or it may be a separate component.

In certain embodiments of the invention, methods allow delivery of agents that promote wound healing. The agent may promote healing by any mechanism. For example and without limitation, the agent may facilitate one or more phases of the wound healing process; prevent infection, including bacterial or viral infection; or alleviate pain or sensitivity.

A variety of growth factors promote wound healing. For example and without limitation, growth factors that promote wound healing include CTGF/CCN2, EGF family members, FGF family members, G-CSF, GM-CSF, HGF, HGH, HIF, histatin, hyaluronan, IGF, IL-1, IL-4, IL-8, KGF, lactoferrin, lysophosphatidic acid, NGF, a PDGF, TGF-P, and VEGF. The EFG family includes 10 members: amphiregulin (AR), betacellulin (BTC), epigen, epiregulin (EPR), heparin- binding EGF-like growth factor (HB-EGF), neuregulin-1 (NRG1), neuregulin-2 (NRG2), neureg- ulin-3 (NRG3), neuregulin-4 (NRG4), or transforming growth factor-a (TGF-a). The FGF family includes 22 members: FGF1, FGF2 (also called basic FGF or bFGF), FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, or FGF23. PDGF exists in three forms: PDGF AA, PDGF AB, and PDGF BB. The TGF-P family includes three forms: TGF-pi, TGF-P2, and TGF-P3.

A variety of agents that prevent infection have been used to treat wounds. For example, and without limitation, the agent may be an antimicrobial, antiviral, antibiotic, antifungal, or antiseptic. Exemplary agents include silver, iodine, chlorhexidine, hydrogen peroxide, lysozyme, peroxidase, defensins, cystatins, thrombospondin, and antibodies. Nitric oxide donors, such as glyceryl trinitrate and nitrite salts, are also useful to prevent infection and promote wound healing.

Diseases, disorders, and conditions

The methods are useful to treat conditions of the GI tract of a subject. The condition may be any disease, disorder, or condition that affects the GI tract.

In some embodiments, the disorder is a disorder of the esophagus, including, but not limited to, esophagitis - (candidal), gastroesophageal reflux disease (gerd); laryngopharyngeal reflux (also known as extraesophageal reflux disease/eerd); rupture (Boerhaave syndrome, Mallory- Weiss syndrome); UES - (Zenker's diverticulum); LES - (Barrett's esophagus); esophageal motility disorder - (nutcracker esophagus, achalasia, diffuse esophageal spasm); esophageal stricture; and megaesophagus.

In some embodiments, the disorder is a disorder of the stomach, including but not limited to gastritis (e.g., atrophic, Menetrier's disease, gastroenteritis); peptic (i.e., gastric) ulcer (e.g., Cushing ulcer, Dieulafoy's lesion); dyspepsia; emesis; pyloric stenosis; achlorhydria; gastropare- sis; gastroptosis; portal hypertensive gastropathy; gastric antral vascular ectasia; gastric dumping syndrome; and human mullular fibrillation syndrome (HMFS).

In some embodiments, the disorder is a disorder of the small intestine, including but not limited to, enteritis (duodenitis, jejuni tis, ileitis); peptic (duodenal) ulcer (curling's ulcer); malabsorption: celiac; tropical sprue; blind loop syndrome; Whipple's; short bowel syndrome; steatorrhea; milroy disease In some embodiments, the disorder is a disorder of the small intestine, including but not limited to, both large intestine and small intestine enterocolitis (necrotizing); inflammatory bowel disease (IBD); Crohn's disease; vascular; abdominal angina; mesenteric ischemia; angiodysplasia; bowel obstruction: ileus; intussusception; volvulus; fecal impaction; constipation; and diarrhea.

In some embodiments, the disorder is a disorder of the small intestine, including but not limited to, accessory digestive glands disease; liver hepatitis (viral hepatitis, autoimmune hepatitis, alcoholic hepatitis); cirrhosis (PBC); fatty liver (Nash); vascular (hepatic veno-occlusive disease, portal hypertension, nutmeg liver); alcoholic liver disease; liver failure (hepatic encephalopathy, acute liver failure); liver abscess (pyogenic, amoebic); hepatorenal syndrome; peliosis hepatis; hemochromatosis; and Wilson's disease.

In some embodiments, the disorder is a disorder of the pancreas, including, but not limited to, pancreas pancreatitis (acute, chronic, hereditary); pancreatic pseudocyst; and exocrine pancreatic insufficiency.

In some embodiments, the disorder is a disorder of the large intestine, including but not limited to, appendicitis; colitis (pseudomembranous, ulcerative, ischemic, microscopic, collagenous, lymphocytic); functional colonic disease (IBS, intestinal pseudoobstruction/ogilvie syndrome); megacolon/toxic megacolon; diverticulitis; and diverticulosis. Tn some embodiments, the disorder is a disorder of the large intestine, including but not limited to, gall bladder and bile ducts, cholecystitis; gallstones/cholecystolithiasis; cholesterolosis; Rokitansky-Aschoff sinuses; postcholecystectomy syndrome cholangitis (PSC, ascending); cho- lestasis/Mirizzi's syndrome; biliary fistula; haemobilia; and gallstones/cholelithiasis. In some embodiments, the disorder is a disorder of the common bile duct (including choledocholithiasis, biliary dyskinesia).

Other disorders which can be treated with the methods and devices included herein include acute and chronic immune and autoimmune pathologies, such as systemic lupus erythematosus (SLE), rheumatoid arthritis, thyroidosis, graft versus host disease, scleroderma, diabetes mellitus, Graves' disease, Beschet' s disease; inflammatory diseases, such as chronic inflammatory pathologies and vascular inflammatory pathologies, including chronic inflammatory pathologies such as sarcoidosis, chronic inflammatory bowel disease, ulcerative colitis, and Crohn's pathology and vascular inflammatory pathologies, such as, but not limited to, disseminated intravascular coagulation, atherosclerosis, giant cell arteritis and Kawasaki's pathology; malignant pathologies involving tumors or other malignancies, such as, but not limited to leukemias (acute, chronic myelocytic, chronic lymphocytic and/or myelodyspastic syndrome); lymphomas (Hodgkin's and non-Hodg- kin's lymphomas, such as malignant lymphomas (Burkitt's lymphoma or Mycosis fungoides)); carcinomas (such as colon carcinoma) and metastases thereof; cancer-related angiogenesis; infantile haemangiomas; and infections, including, but not limited to, sepsis syndrome, cachexia, circulatory collapse and shock resulting from acute or chronic bacterial infection, acute and chronic parasitic and/or infectious diseases, bacterial, viral or fungal, such as a HIV, AIDS (including symptoms of cachexia, autoimmune disorders, AIDS dementia complex and infections).

Other disorders which can be treated with the methods and devices included herein include acute and chronic immune and autoimmune pathologies, inflammatory diseases, infections and malignant pathologies involving, e.g., tumors or other malignancies.

The subject suffering from the GI condition may be any type of subject, such as an animal, for example, a mammal, for example, a human. Incorporation by Reference

References and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web contents, have been made throughout this disclosure. All such documents are hereby incorporated herein by reference in their entirety for all purposes.

Equivalents

Various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the full contents of this document, including references to the scientific and patent literature cited herein. The subject matter herein contains important information, exemplification, and guidance that can be adapted to the practice of this invention in its various embodiments and equivalents thereof.