Compositions and methods for targeted delivery of therapeutics using carriers

Abstract

The present invention provides novel compositions and methods for targeted delivery of therapeutics. In particular, the invention provides compositions comprising a plurality of carriers, such as microbubbles, wherein at least one active agent is associated or co-administered with the plurality of carriers for delivery to a target site, e.g., an organ, a tissue, or a tumor site, in a subject. The present invention also provides methods for treating a disease or condition, methods of targeted delivery of an active agent, e.g., to a target site, in a subject, using the carriers based compositions of the invention. The present invention further provides apparatus, devices and methods for preparing the compositions of the present invention.

1 . A composition comprising a plurality of carriers, wherein at least one active agent is associated with the plurality of carriers for delivery to a subject,
 (a) wherein the level of the at least one active agent associated with the plurality of carriers is lower than the level of the active agent required to achieve a therapeutic effect when administered without being associated with carriers, and/or   (b) wherein the carriers comprise a shell and a core, optionally wherein
 (i) the shell comprises a lipid comprising 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC); 
 (ii) the core comprises a perfluorohexane gas and/or nitrogen; 
 (iii) the shell comprises a lipid, and the core comprises a perfluorohexane gas and/or nitrogen; and/or 
 (iv) the shell comprises a lipid comprising 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and the core comprises a gas comprising a perfluorocarbon gas, nitrogen, or combination thereof. 
   
     
     
         2 . The composition of  claim 1 , wherein the level of the at least one active agent associated with the plurality of carriers is lower than the level of the active agent required to achieve a therapeutic effect when administered without being associated with carriers and by systemic administration, optionally
 (i) wherein the level of the at least one active agent associated with the plurality of carriers is lower than the level of the active agent required to achieve a substantially equivalent therapeutic effect when administered without being associated with carriers,
 optionally wherein the substantially equivalent therapeutic effect is about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% of the therapeutic effect of the active agent when administered without being associated with carriers; or 
   (ii) wherein the level of the at least one active agent associated with the plurality of carriers is about 0.001% to about 99%, about 0.001% to about 75%, about 0.001% to about 50%, about 0.005% to about 50%, about 0.005% to about 25%, or about 0.0001% to about 25% of the level of the active agent required to achieve a therapeutic effect, optionally, a substantially equivalent therapeutic effect, when administered without being associated with carriers; and/or   (iii) wherein the level of the at least one active agent associated with the plurality of carriers is about 0.001% to about 0.005%, about 0.005% to about 0.1%, about 0.005% to about 10%, about 0.005% to about 20%, about 10% to about 50%, or about 50% to about 99% of the level of the active agent required to achieve a therapeutic effect, optionally a substantially equivalent therapeutic effect, when administered without being associated with carriers.   
     
     
         3 .- 4 . (canceled) 
     
     
         5 . The composition of  claim 1 , wherein the carrier comprises a shell and a core, optionally wherein
 (a) (i) the shell comprises a lipid, a polymer, a lipopolymer, a protein, or combination thereof,
 optionally wherein the lipid is selected from the group consisting of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 4-dimethylaminochalcone (DMAC), dipalmitoylphosphatidylcholine (DPPC), 1,2-distearyol-sn-glycero-3-phosphocoline (DSPC), and 1,2-distearyol-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)2000] (DSPE-PEG2000), and/or 
 optionally wherein the lipid is DMPC; 
    (ii) the core comprises a gas;    (iii) the core comprises nitrogen, air, a perfluorocarbon gas, or combination thereof,
 optionally wherein the perfluorocarbon gas is selected from the group consisting of perfluorohexane, sulfur hexafluoride, perfluoromethane, perfluoroethane, perfluoropropane, perfluorobutane, and perfluoropentane, and/or 
 optionally wherein the perfluorocarbon gas is perfluorohexane; and/or 
    (iv) the core comprises a combination of perfluorohexane and nitrogen gas,   (b) wherein the carrier is selected from the group consisting of a microbubble, a nanobubble, a nanoparticle, a nanodroplet, a micelle, a liposome, an exosome, a cell, and a virus, and/or   (c) wherein the carrier is a microbubble, optionally selected from the group consisting of Levovist™, IMAGENT®, Optison®, Sonazoid®, BR38, and SonoVue®.   
     
     
         6 .- 17 . (canceled) 
     
     
         18 . The composition of  claim 1 , wherein the at least one active agent is associated with the plurality of carriers by being encapsulated within the plurality of carriers,
 optionally wherein the at least one active agent is associated with the plurality of carriers by binding to the interior or exterior surface of the plurality of carriers.   
     
     
         19 .- 21 . (canceled) 
     
     
         22 . The composition of  claim 1 , wherein administration of the composition results in a reduced systemic toxicity, as compared to administration of a composition of the same level of active agent without being associated with carriers,
 optionally wherein administration of the composition results in a reduced systemic toxicity, as compared to administration of a composition of the active agent without being associated with carriers required to achieve a substantially equivalent therapeutic effect.   
     
     
         23 . (canceled) 
     
     
         24 . The composition of  claim 1 , wherein the level of the at least one active agent associated with the plurality of carriers elicits an immunostimulatory effect,
 optionally wherein the level of the at least one active agent associated with the plurality of carriers does not elicit an immunosuppressive effect.   
     
     
         25 . (canceled) 
     
     
         26 . The composition of  claim 1 , wherein the at least one active agent is selected from the group consisting of a protein, an antibody, a small molecule, a virus, an antibiotic, a radionuclide, a peptide, a nucleic acid, a gene, a vector or a plasmid encoding a gene, and a component of a gene editing system, optionally wherein
 (a) the at least one active agent comprises a cytokine, and/or a vector or plasmid encoding a gene encoding the cytokine, optionally wherein
 (i) the cytokine is selected from a group consisting of IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21, IL-23, IL-27, interferon, GM-CSF and TNF-alpha; and/or 
 (ii) the cytokine is IL-2,
 optionally wherein the level of IL-2 associated with the plurality of carriers is about 0.01 pg/mL to about 10 pg/mL, about 10 pg/mL to about 100 pg/mL, about 100 pg/mL to about 100 ug/mL, about 100 pg/mL to about 100 ng/mL, about 100 ng/mL to about 1 μg/mL, about 1 μg/ml to about 10 μg/mL, about 10 μg/mL to about 100 μg/mL, about 200 ng/mL to about 500 ng/mL, about 500 ng/mL to about 2 μg/mL, or about 2 μg/mL to about 5 μg/mL; and/or 
 
   (b) the at least one active agent comprises a monoclonal antibody, and/or a vector or plasmid comprising a gene encoding the monoclonal antibody, optionally wherein
 (i) the monoclonal antibody is selected from the group consisting of bevacizumab, pembrolizumab, nivolumab, cemiplimab, durvalumab, atezolizumab, avelumab, ipilimumab, rituximab, cetuximab and trastuzumab; 
 (ii) the monoclonal antibody is bevacizumab,
 optionally wherein the level of bevacizumab associated with the plurality of carriers is about 0.01 pg/mL to about 10 pg/mL, about 1 pg/mL to about 25 mg/mL, about 1 ng/mL to about 25 mg/mL, about 1 μg/mL to about 25 mg/mL, about 1 pg/mL to about 100 pg/mL, about 100 pg/mL to about 500 pg/mL, about 500 pg/mL to about 1 ng/mL, about 1 ng/mL to about 100 ng/mL, about 100 ng/mL to about 500 ng/mL, about 500 ng/mL to about 1 μg/mL, about 1 μg/mL to about 100 μg/mL, about 100 μg/mL to about 500 μg/mL, about 500 μg/mL to about 1 mg/mL, about 1 mg/mL to about 5 mg/mL, about 5 mg/mL to about 10 mg/mL, about 10 mg/mL to about 25 mg/mL, about 100 μg/mL to about 250 μg/mL, about 250 μg/mL to about 2.5 mg/mL, or about 2.5 mg/mL to about 10 mg/mL; and/or 
 
 (iii) the monoclonal antibody is pembrolizumab,
 optionally wherein the level of pembrolizumab associated with the plurality of carriers is about 0.01 pg/mL to about 10 pg/mL, about 1 pg/mL to about 25 mg/mL, about 1 ng/mL to about 25 mg/mL, about 1 μg/mL to about 25 mg/mL, about 1 pg/mL to about 100 pg/mL, about 100 pg/mL to about 500 pg/mL, about 500 pg/mL to about 1 ng/mL, about 1 ng/mL to about 100 ng/mL, about 100 ng/mL to about 500 ng/mL, about 500 ng/mL to about 1 μg/mL, about 1 μg/mL to about 100 μg/mL, about 100 μg/mL to about 500 μg/mL, about 500 μg/mL to about 1 mg/mL, about 1 mg/mL to about 5 mg/mL, about 5 mg/mL to about 10 mg/mL, about 10 mg/mL to about 25 mg/mL, about 100 μg/mL to about 250 μg/mL, about 250 μg/mL to about 2.5 mg/mL, or about 2.5 mg/mL to about 10 mg/mL. 
 
   
     
     
         27 .- 36 . (canceled) 
     
     
         37 . The composition of  claim 1 , wherein the at least one active agent comprises one or more components of a gene editing system, optionally
 wherein the at least one active agent comprises one or more components of a CRISPR system, and/or   wherein the at least one active agent comprises a cDNA and/or mRNA encoding a Cas9 protein, a guide RNA, and/or a ribonucleoprotein (RNP) complex.   
     
     
         38 .- 39 . (canceled) 
     
     
         40 . The composition of  claim 26 , wherein the virus is an oncolytic virus,
 wherein the oncolytic virus is selected from the group consisting of adenovirus, adeno-associated virus, herpes virus, poliovirus, measles virus, polioviruses, poxvirus, Newcastle disease virus, reovirus, coxsackievirus, vesicular stomatitis virus, Zika virus, RNA virus, and DNA virus.   
     
     
         41 . (canceled) 
     
     
         42 . The composition of  claim 1 , wherein the composition comprises a viral vector comprising a gene encoding the active agent. 
     
     
         43 .- 44 . (canceled) 
     
     
         45 . The composition of  claim 1 ,
 (i) wherein the composition, or the preparation thereof, is free of an inactivation agent for the active agent, optionally
 wherein the composition, or the preparation thereof, is free of a viral inactivating agent, and/or 
    (ii) wherein the composition is free of a binding agent or a binding ligand.   
     
     
         46 .- 47 . (canceled) 
     
     
         48 . The composition of  claim 1  further comprising a plurality of free lipids, optionally wherein
 (a) the plurality of free lipids comprise at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the total lipid content in the composition; 
 (b) the plurality of free lipids are in a high energy state before reconstitution of the carrier; 
 (c) the plurality of free lipids form a second plurality of carriers upon exposure to an aqueous solution; 
 (d) the plurality of free lipids form a second plurality of carriers comprising a liposome, a lipid sheet, a lipid rod, a lipid disc, or a combination thereof; 
 (e) the plurality of free lipids form a second plurality of carriers comprising liposome, optionally a unilamellar or multilamellar liposome; and/or 
 (f) the plurality of free lipids comprises DMPC. 
 
     
     
         49 .- 51 . (canceled) 
     
     
         52 . The composition of  claim 48 , wherein the at least one active agent is encapsulated in, bound to, associated with or interacted with the second plurality of carriers. 
     
     
         53 .- 54 . (canceled) 
     
     
         55 . The composition of  claim 48 , wherein the first plurality of carriers comprises microbubbles,
 wherein the microbubbles comprise:   (i) a shell comprising a lipid, a polymer, a lipopolymer, a protein, or combination thereof,
 optionally wherein the lipid is DMPC; and/or 
   (ii) a core comprising a gas,
 optionally wherein the core comprises at least one of perfluorohexane and nitrogen gas. 
   
     
     
         56 .- 58 . (canceled) 
     
     
         59 . A method of treating a disease or condition, delivering an active agent to a target site or reducing tumor growth in a subject, the method comprising:
 administering to the subject a composition comprising at least one active agent associated with a plurality of carriers,   (a) wherein the level of the at least one active agent associated with the plurality of carriers is lower than the level of the active agent required to achieve a therapeutic effect when administered without being associated with carriers, and/or   (b) wherein the carriers comprise a shell and a core, optionally wherein
 (i) the shell comprises a lipid comprising 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC); 
 (ii) the core comprises a perfluorohexane gas and/or nitrogen; 
 (iii) the shell comprises a lipid, and the core comprises a perfluorohexane gas and/or nitrogen; and/or 
 (iv) the shell comprises a lipid comprising 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and the core comprises a gas comprising a perfluorocarbon gas, nitrogen, or combination thereof, and 
   applying an ultrasound energy to the subject, thereby treating the disease or condition, delivering the active agent to the target site or reducing tumor growth in the subject.   
     
     
         60 .- 64 . (canceled) 
     
     
         65 . The method of  claim 59 , wherein the target site is an organ, a tissue or a tumor site in the subject. 
     
     
         66 . The method of  claim 59 , further comprising administering to the subject a composition comprising at least one free active agent. 
     
     
         67 . The method of  claim 59 , wherein the composition further comprises a plurality of free lipids, optionally wherein
 (i) the plurality of free lipids comprise at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the total lipid content in the composition;   (ii) the plurality of free lipids are in a high energy state before reconstitution of the carrier;   (iii) the plurality of free lipids form a second plurality of carriers comprising a liposome, a lipid sheet, a lipid rod, a lipid disc, or a combination thereof;   (iv) the plurality of free lipids form a second plurality of carriers comprising a liposome, optionally a unilamellar or multilamellar liposome; and   (v) the plurality of free lipids comprises DMPC.   
     
     
         68 .- 69 . (canceled) 
     
     
         70 . The method of  claim 67 , further comprising exposing the composition to an aqueous solution prior to administration to the subject, optionally wherein
 (i) the plurality of free lipids form a second plurality of carriers upon exposure to the aqueous solution,
 wherein the at least one active agent is encapsulated in, bound to, associated with or interacted with the second plurality of carriers upon exposure to the aqueous solution. 
   
     
     
         71 .- 74 . (canceled) 
     
     
         75 . A method of treating a disease or condition, delivering an active agent to a target site or reducing tumor growth in a subject, the method comprising:
 administering to the subject a first composition comprising a plurality of carriers, optionally wherein the carriers comprise a shell and a core, optionally wherein,
 (i) the shell comprises a lipid comprising 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC); 
 (ii) the core comprises a perfluorohexane gas and/or nitrogen; 
 (iii) the shell comprises a lipid, and the core comprises a perfluorohexane gas and/or nitrogen; and/or 
 (iv) the shell comprises a lipid comprising 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and the core comprises a gas comprising a perfluorocarbon gas, nitrogen, or combination thereof; and 
   administering to the subject a second composition comprising at least one active agent, wherein the level of the at least one active agent co-administered with the plurality of carriers is lower than the level of the active agent required to achieve a therapeutic effect when administered without being co-administered with carriers, and   applying an ultrasound energy to the subject, thereby treating the disease or condition, delivering the active agent to the target site or reducing tumor growth in the subject.   
     
     
         76 .- 80 . (canceled) 
     
     
         81 . The method of  claim 75 , wherein the target site is an organ, a tissue or a tumor site in the subject. 
     
     
         82 . The method of  claim 75 , wherein the first composition further comprises a plurality of free lipids, optionally wherein
 (i) the plurality of free lipids comprise at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the total lipid content in the composition,   (ii) the plurality of free lipids are in a high energy state before reconstitution of the carrier,   (iii) the plurality of free lipids form a second plurality of carriers comprising a liposome, a lipid sheet, a lipid rod, a lipid disc, or a combination thereof,   (iv) the plurality of free lipids form a second plurality of carriers comprising a liposome, optionally a unilamellar or multilamellar liposome, and   (v) the plurality of free lipids comprises DMPC.   
     
     
         83 .- 84 . (canceled) 
     
     
         85 . The method of  claim 82 , further comprising exposing the composition to an aqueous solution prior to administration to the subject,
 wherein the plurality of free lipids form a second plurality of carriers upon exposure to the aqueous solution.   
     
     
         86 .- 89 . (canceled) 
     
     
         90 . The method of  claim 59 , wherein the level of the at least one active agent associated with the plurality of carriers is lower than the level of the active agent required to achieve a therapeutic effect when administered without being associated with carriers by systemic administration,
 optionally wherein the level of the at least one active agent associated with the plurality of carriers is lower than the level of the active agent required to achieve a substantially equivalent therapeutic effect when administered without being associated with carriers,
 wherein the substantially equivalent therapeutic effect is about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% of the therapeutic effect of the active agent when administered without being associated with carriers, 
   optionally wherein the level of the at least one active agent associated with the plurality of carriers is about 0.001% to about 99%, about 0.001% to about 75%, about 0.001% to about 50%, or about 0.001% to about 25% of the level of the active agent required to achieve a therapeutic effect, optionally, a substantially equivalent therapeutic effect, when administered without being associated or co-administered with carriers, and/or   optionally wherein the level of the at least one active agent associated with the plurality of carriers is about 0.001% to about 0.1%, about 0.1% to about 10%, about 0.1% to about 20%, about 10% to about 50%, or about 50% to about 99% of the level of the active agent required to achieve a therapeutic effect, optionally a substantially equivalent therapeutic effect, when administered without being associated or co-administered with carriers.   
     
     
         91 .- 92 . (canceled) 
     
     
         93 . The method of  claim 59 , wherein applying the ultrasound energy induces cavitation of the carriers and/or enhances delivery of the active agent within the subject. 
     
     
         94 .- 95 . (canceled) 
     
     
         96 . The method of  claim 59 , wherein administration of the composition results in a reduced systemic toxicity,
 (i) as compared to administration of a composition of the same level of active agent without being associated with carriers, or   (ii) as compared to administration of a composition of the active agent without being associated with carriers required to achieve a substantially equivalent therapeutic effect.   
     
     
         97 . (canceled) 
     
     
         98 . The method of  claim 59 , wherein the level of the at least one active agent associated or co-administered with the plurality of carriers elicits an immunostimulatory effect, and/or
 wherein the level of the at least one active agent associated with the plurality of carriers does not elicit an immunosuppressive effect.   
     
     
         99 . (canceled) 
     
     
         100 . The method of  claim 59 , wherein the at least one active agent is selected from a group consisting of a protein, an antibody, a small molecule, a virus, an antibiotic, a radionuclide, a peptide, a nucleic acid, a gene, a vector or a plasmid encoding a gene, and a component of a gene editing system, optionally wherein
 (a) the at least one active agent comprises a cytokine, and/or a vector or plasmid encoding a gene encoding the cytokine, optionally wherein
 (i) the cytokine is selected from a group consisting of IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21, IL-23, IL-27, interferon, GM-CSF and TNF-alpha; and/or 
 (ii) the cytokine is IL-2,
 wherein the level of IL-2 associated with the plurality of carriers is about 0.01 pg/mL to about 10 pg/mL, about 10 pg/mL to about 100 pg/mL, about 100 pg/mL to about 100 ug/mL, about 100 pg/mL to about 100 ng/mL, about 100 ng/mL to about 1 μg/mL, about 1 μg/ml to about 10 μg/mL, about 10 μg/mL to about 100 μg/mL, about 200 ng/mL to about 500 ng/mL, about 500 ng/mL to about 2 μg/mL, or about 2 μg/mL to about 5 ug/mL; and/or 
 
   (b) the at least one active agent comprises a monoclonal antibody, and/or a vector or plasmid comprising a gene encoding the monoclonal antibody, optionally wherein
 (i) the monoclonal antibody is selected from the group consisting of bevacizumab, pembrolizumab, nivolumab, cemiplimab, durvalumab, atezolizumab, avelumab, ipilimumab, rituximab, cetuximab and trastuzumab; 
 (ii) the monoclonal antibody is bevacizumab,
 wherein the level of bevacizumab associated with the plurality of carriers is about 0.01 pg/mL to about 10 pg/mL, about 1 pg/mL to about 25 mg/mL, about 1 ng/mL to about 25 mg/mL, about 1 μg/mL to about 25 mg/mL, about 1 pg/mL to about 100 pg/mL, about 100 pg/mL to about 500 pg/mL, about 500 pg/mL to about 1 ng/mL, about 1 ng/mL to about 100 ng/mL, about 100 ng/mL to about 500 ng/mL, about 500 ng/mL to about 1 μg/mL, about 1 μg/mL to about 100 μg/mL, about 100 μg/mL to about 500 μg/mL, about 500 μg/mL to about 1 mg/mL, about 1 mg/mL to about 5 mg/mL, about 5 mg/mL to about 10 mg/mL, about 10 mg/mL to about 25 mg/mL, about 100 μg/mL to about 250 μg/mL, about 250 μg/mL to about 2.5 mg/mL, or about 2.5 mg/mL to about 10 mg/mL; and/or 
 
 (iii) the monoclonal antibody is pembrolizumab,
 wherein the level of pembrolizumab associated with the plurality of carriers is about 0.01 pg/mL to about 10 pg/mL, about 1 pg/mL to about 25 mg/mL, about 1 ng/mL to about 25 mg/mL, about 1 μg/mL to about 25 mg/mL, about 1 pg/mL to about 100 pg/mL, about 100 pg/mL to about 500 pg/mL, about 500 pg/mL to about 1 ng/mL, about 1 ng/mL to about 100 ng/mL, about 100 ng/mL to about 500 ng/mL, about 500 ng/mL to about 1 μg/mL, about 1 μg/mL to about 100 μg/mL, about 100 μg/mL to about 500 μg/mL, about 500 μg/mL to about 1 mg/mL, about 1 mg/mL to about 5 mg/mL, about 5 mg/mL to about 10 mg/mL, about 10 mg/mL to about 25 mg/mL, about 100 μg/mL to about 250 μg/mL, about 250 μg/mL to about 2.5 mg/mL, or about 2.5 mg/mL to about 10 mg/mL. 
 
   
     
     
         101 .- 110 . (canceled) 
     
     
         111 . The method of  claim 59 , wherein the at least one active agent comprises one or more components of a gene editing system,
 optionally wherein the at least one active agent comprises one or more components of a CRISPR system, and/or   optionally wherein the at least one active agent comprises a cDNA and/or mRNA encoding a Cas9 protein, a guide RNA, and/or a ribonucleoprotein (RNP) complex.   
     
     
         112 .- 113 . (canceled) 
     
     
         114 . The method of  claim 100 , further wherein the virus is an oncolytic virus,
 optionally wherein the oncolytic virus is selected from the group consisting of adenovirus, adeno-associated virus, herpes virus, poliovirus, measles virus, polioviruses, poxvirus, Newcastle disease virus, reovirus, coxsackievirus, vesicular stomatitis virus, Zika virus, RNA virus, and DNA virus.   
     
     
         115 . (canceled) 
     
     
         116 . The method of  claim 59 , wherein the composition comprises a viral vector comprising a gene encoding the active agent. 
     
     
         117 . The method of  claim 59 , wherein the at least one active agent is associated with the plurality of carriers by being encapsulated within the plurality of carriers,
 optionally wherein the at least one active agent is associated with the plurality of carriers by binding to the exterior or interior surface of the plurality of carriers.   
     
     
         118 . (canceled) 
     
     
         119 . The method of  claim 59 , wherein the carrier is selected from the group consisting of a microbubble, a nanobubble, a nanoparticle, a nanodroplet, a micelle, a liposome, an exosome, a cell, and a virus,
 optionally wherein the carrier is a microbubble, optionally selected from the group consisting of Levovist™, IMAGENT®, Optison®, Sonazoid®, BR38, and SonoVue®.   
     
     
         120 .- 125 . (canceled) 
     
     
         126 . A method for reducing systemic toxicity of an active agent or reducing innate immune response against an active agent upon administration into a subject, the method comprising:
 (i) providing a composition comprising a plurality of carriers,
 associating the active agent with the plurality of carriers, 
 administering the plurality of carriers associated with the active agent to the subject, and 
 applying an ultrasound energy to the subject, thereby reducing the systemic toxicity or the innate immune response of the active agent; or 
   (ii) administering to the subject a first composition comprising a plurality of carriers,
 administering to the subject a second composition comprising the active agent, and 
 applying an ultrasound energy to the subject, thereby reducing the systemic toxicity of or reducing the innate immune response against the active agent. 
   
     
     
         127 .- 129 . (canceled) 
     
     
         130 . The method of  claim 126 , wherein the carriers comprise a shell and a core, optionally wherein
 (i) the shell comprises a lipid comprising 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC);   (ii) the core comprises a perfluorohexane gas and/or nitrogen;   (iii) the shell comprises a lipid, and the core comprises a perfluorohexane gas and/or nitrogen; and/or   (iv) the shell comprises a lipid comprising 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and the core comprises a gas comprising a perfluorocarbon gas, nitrogen, or combination thereof.   
     
     
         131 . The method of  claim 126 , wherein associating the active agent with the plurality of carriers comprises encapsulating the active agent within the plurality of carriers or binding the active agent to the interior or exterior surface of the plurality of carriers. 
     
     
         132 .- 167 . (canceled)