COMMUNICATIONUS009731012B2 0100 (12 ) United States Patent (10 ) Patent No. : US 9 , 731, 012 B2 Arany et al. ( 45) Date of Patent: Aug . 15 , 2017 ( 54 ) LASER -ACTUATED THERAPEUTIC .. .. .. .. .. .. .. .. .. .. .. .. 424 / 400 ; 514 / 1 . 1 , 8 . 9 NANOPARTICLES See application file for complete search history . (71 ) Applicant : President and Fellows of Harvard (56 ) References Cited College, Cambridge , MA (US ) U . S . PATENT DOCUMENTS (72 ) Inventors: Praveen Arany , Cambridge , MA (US ) ; 2010 /0183728 Al * 7 /2010 Desai .. .. .. A61K 9 /0019 David J . Mooney , Sudbury , MA (US ) 424 /489 2010/ 0196490 A1 * 8 / 2010 Desai .. A61K 9 /0019 (73 ) Assignee : President and Fellows of Harvard 424 / 489 College , Cambridge , MA (US ) 2010 /0226996 A1* 9 / 2010 Desai A61K 9 /0019 424 /499 2012 /0076862 A1* 3 /2012 Desai . .. .. .. .. A61K 31 / 337 ( * ) Notice : Subject to any disclaimer , the term of this 424 / 491 patent is extended or adjusted under 35 2013 /0231287 A1 * 9 /2013 Nacharaju . .. .. A61K 47 /48215 U . S . C . 154 ( b ) by 98 days. 514 / 15 . 2 (21 ) Appl. No .: 14 / 389 , 136 OTHER PUBLICATIONS (22 ) PCT Filed : Mar . 12 , 2013 Arany et al. , Wound Repair Regen , 15 ( 6 ) :866 - 74 . (2007 ) “ Activa tion of latent TGF- betal by low -power laser in vitro correlates with ( 86 ) PCT No. : PCT/ US2013 / 030493 increased TGF -betal levels in laser -enhanced oral wound healing .” Arany , P . R ., Proceedings of Light- Activated Tissue Regeneration $ 371 ( c ) ( 1 ) , and Therapy Conference Lecture Notes in Electrical Engineering ( 2 ) Date : Sep . 29 , 2014 vol . 12 , pp . 207 - 212 . ( 2008 ) “ Photobiomodulation by Low Power Laser Irradiation Involves Activation of Latent TGF -B1 ” . (87 ) PCT Pub. No. : W02013 / 148158 Jeong et al ., Theranostics , 1 : 230 -239 , ( 2011) . “ Photosensitizer Conjugated human serum albumin nanoparticles for effective PCT Pub . Date : Oct . 3 , 2013 photodynamic therapy . ” Kratz et al. , J Control Release 132 ( 3 ) : 171 -83 . ( 2008 ) . “ Albumin as (65 ) Prior Publication Data a drug carrier : design of prodrugs , drug conjugates and nanoparticles .” US 2015 /0065685 A1 Mar. 5 , 2015 Lu et al. , J Control Release , 107 ( 3 ) :428 -48 . ( 2005 ) " Cationic albumin - conjugated pegylated nanoparticles as novel drug carrier Related U . S . Application Data for brain delivery. ” ( 60 ) Provisional application No . 61/ 617 ,822 , filed on Mar . 30 , 2012 . * cited by examiner (51 ) Int. CI. Primary Examiner — David Lukton A61K 47 / 48 ( 2006 .01 ) (74 ) Attorney, Agent, or Firm - McCarter & English , A6IK 41/ 00 ( 2006 . 01 ) LLP ; Maria Laccotripe Zacharakis ; Deborah L . Nagle A61K 38 / 18 ( 2006 .01 ) A61K 38 /00 ( 2006 .01 ) (57 ) ABSTRACT (52 ) U . S . CI. The invention provides compositions and methods for laser CPC . .. .. A61K 41/ 0042 ( 2013 .01 ) ; A61K 38 / 1841 actuated drug delivery . Compositions comprise serum albu (2013 . 01 ) ; A61K 47 /48284 (2013 .01 ) ; A61K min based particles conjugated with therapeutic agents 47/ 48884 ( 2013 .01 ) ; A61K 38 / 00 (2013 .01 ) which cab become bioavailable upon actuation of the par (58 ) Field of Classification Search ticles by light , e . g . low power laser . CPC .. A61K 47 /48884 ; A61K 41/ 0042 ; A61K 38 / 00 ; A61K 38 / 1841 ; A61K 47/ 48284 17 Claims, 24 Drawing Sheets atent Aug . 15 , 2017 Sheet 1 of 24 US 9 ,731 , 012 B2 mgWater • BSA CONTROL ABSA LASER hobi A mDeg N2 teelDDIDDIWDLIDATI XXX << < < < < < << < << < < < < < < << < < < < < < < << < < ** * * * * 3 modae con 190 200 210 220 230 240 250 260 WAVELENGTH (nm ) A FIG . 1 LASER 9. 9903 INFLAMMATION 80FIG . 2 U . S . Patent Aug . 15, 2017 Sheet 2 of 24 US 9 ,731 , 012 B2 HSA DRUGHSACONJUGATE HSA ANTIBODY-BINDING COMPLEXES >=PEPTIDE DRUG=O ODXC drugsasconjugates,complexesornanoparticles.FiguetakenfromKratzetal2011[1] Applicationsofalbuminasadrugcarrierfordeveloped-peptide,antibodyorbased FIG.3 HSA=HUMAN SERUMALBUMIN HSA Albuminasadrugcarrier HSAODvd DRUGHSACOMPLEX DRUGHSANANOPARTICLES o=PEPTIDE O=DRUG U . S . Patent Aug . 15, 2017 Sheet 3 of 24 US 9 ,731 , 012 B2 EFFECTORFUNCTIONS EFFECTORFUNCTION CHEMOTAXIS #SURVIVAL OPROLIFERATION ODIFFERENTIATION GENERATIONINTHEPERIPHERY EFFECTORFUNCTIONS I FoxP3EXPRESSION CHEMOTAXIS TCELL OHOMEOSTASIS 70°°°\ PMN Treg FOR EFFECTORFUNCTIONS EO NKCELL HOMEOSTASISOCYTOKINEPRODUCTION CYTOTOXICITY FIG.4 CHEMOTAXIS 008 BCELL MO/PRESENTATION)ANTIGENO DO SWITCHINGIgA EFFECTORFUNCTIONS zn OPROLIFERATION OACTIVATION OSURVIVAL +CHEMOTAXISOSCAVENGERFUNCTION LANGERHANSCELLDEVELOPMENT ANTIGENPRESENTATION FUNCTIONSOFTGF-B OMATURATION OANTIGENPRESENTATION STIMULATES?MAINTAINS OINHIBITS UNCLEAR U . S . Patent Aug . 15, 2017 Sheet 4 of 24 US 9 , 731, 012 B2 - 1 . , ' . : / FIG . 5 INITIAL CONDITIONIN BSABSA NPNP LOADING FRACTION 1ST ( CONJUGATION SOLUTION ) CENTRIFUGE TGF- B1 - ( LUTE - - ( UNCONJUGATED FRACTION ) ACTIVATION CONTROL LASER BOUND FRACTION ACTIVATION (CONTROL )( LASER H202/ 0TT (BOUND BIOAVAILABILITY ) FIG . 6 U . S . Patent Aug . 15 , 2017 Sheet 5 of 24 US 9 . 731 , 012 B2 4 1 ? LaLau 1 . : FIG.7 . .”: . ? , " : s : : * : : : HLP ' rik . ru U . S . Patent Aug . 15, 2017 Sheet 6 of 24 US 9 ,731 , 012 B2 ?? ? 0PTICALDENSITY(570nm) ? ? ? ? mg 7? _ 30 _ 80 _ 80 120 150 TIME ( IN) FIG. 8 - * - ' . ' * . " . ' . ' , ' , ' , ' , ' . ' . FIG. 9 atent Aug . 15 , 2017 Sheet 7 of 24 US 9 , 731 , 012 B2 ??? FIG.11D FIG.11c ?????? FIG.10 FIG.11B ??FIG.11A U . S . Patent Aug . 15, 2017 -Sheet 8 of -24 US 9 ,731 , 012 B2 INTENSITY(%) ttt -- w 7. mm LLLLLLLLLLL kuLL Ik LILI OL 100 0001 00001 SIZE (d . nm ) FIG . 12A INTENSITY(%)2 WILL - 10 001 0001 00001 SIZE ( d. nm )Oh . .. oo .. FIG . 12B INTENSITY(%) 5.749- .. .. .. .. .. .vara LLLL . .. .. .. .. .. .. .. L 100 0001 00001 SIZE (d .nm ) FIG . 120 atent Aug . 15 , 2017 Sheet 9 of 24 US 9 ,731 , 012 B2 100 um FIG . 13A PROFILE EXTRACTION 1 www - * - ultimatethatthetitleoftwoother - ! ALTITUDE(nm) * than * - - - - - the ASUSUNANISASIUMISMASNENUSTASSNIUS - -77777777777777777777F7777777 - siwww - wwwwwwwwwwwww il why DISTANCE: 789. 68 um . MINIMUM ALTITUDE: - 3. 3 nm - - - - - - - - - - - - - - - - - - - MAXIMUM ALTITUDE: 31. 0 nm AVERAGE ALTITUDE : 2 . 9 nm MTWITTTTTTTTTTTTTTTTTT0 100 200 300 400 500 600 700 DISTANCE (um ) FIG . 13B FIG . 14 U . S . Patent Aug . 15, 2017 Sheet 10 of 24 US 9 ,731 ,012 B2 CARBON 80% SULFUR FIG.15B 8% SODIUM y CHLORINE 11% .111 IM 52% " 7. CHLORINE W SULFUR 0% FIG.15A SODIUM 27% ** 21% * CARBON U . S . Patent Aug . 15, 2017 Sheet 11 of 24 US 9 ,731 , 012 B2 MAANAAAAAAAAHHH FIG . 16A FIG . 16B FIG . 16C * UM)CYSTEINE(FREE =???? CONTROL H202 BSA FIG . 17 U . S . Patent Aug . 15 , 2017 Sheet 12 of 24 US 9 ,731 , 012 B2 * FREECYSTEINES(UM) q????? CONTROL H202 CONTROL BSABC NPS H202 FIG . 18 FOLDINCREASEOVERCONTROL(RFU) 105 CONTROL LASER BSABSA MO NPS LASER FIG . 19A * FOLDINCREASEOVERCONTROL(UM) po-op CONTROL LASER BSA NPS FIG . 19B U . S . Patent Aug . 15, 2017 Sheet 13 of 24 US 9 ,731 , 012 B2 INTENSITY(%) 27.42 TT .. .. TITIITTIT TTTTTTTTTTI 10001000 1000010000 FIG SIZE. ( d.20A nm ) INTENSITY(%) TTC TTTTTTT TTTTTTT TTTTTTT TTTTT T TTTTTTT 10 100100 1000100001000 10000 SIZE (d . nm ) FIG . 20B INTENSITY(%) 1 27.9 TIT TTTTT .. quam TIIM 10 100 1000 10000 SIZE ( d .nm ) FIG . 200 INTENSITY(%) 27.9?š TTTTTTTTTTTTTTTT TTTTTTT . 100 1000 10000 SIZE ( d .nm ) FIG . 20D U . S . Patent Aug . 15, 2017 Sheet 14 of 24 US 9 ,731 , 012 B2 FOLDINCREASE(DECREASE) OVERCONTROL(RFUS) * -02 CONTROL CONJUGATED BSA - EMBEDDED BSA TGF- b1 NP TGF- b1 NP FIG . 21 FOLDINCREASEOVERCONTROL(RFUS) CONTROL LASER CONJUGATED BSA - TGF- b1 NPS FIG . 22 U . S . Patent Aug . 15, 2017 Sheet 15 of 24 US 9 ,731 , 012 B2 BSA NPS BSA -TGF -B1 NPS CONTROL LASER CONTROL LASER FIG . 23 TGFb-1(pg/mL) § CONTROL LASER CONJUGATED BSA - TGFb - 1 NANOPARTICLES FIG . 24 na manatent Augwo . 15 , 2017 menSheet 16 of 24 US 9 ,731 , 012 B2 FOLDINCREASEOVERCONTROLS (LUCIFERASEACTIVITY) anon CONTROL 3J/ cm2 30J / cm2 LASER CONJUGATED BSA - TGFb- 1 NANOPARTICLES buFIG . 25 12000 10000 8000 LUCIFERASEACTIVITY 6000 4000 D 2000 Do ZŠ Š 10 TGF- B1 CONCENTRATION (ng /mL ) FIG . 26 U . S . Patent Aug. 15 , 2017 Sheet 17 of 24 US 9 ,731 , 012 B2 SEAP(WU/RFU*106 Incopara CONTROL LPSLPS LPS + TGF -b1 FIG . 27 SEAP(WU/RFU*106) qon# CONTROL LPS LASER + LPS CONJUGATED BSA - TGF- b1 NPS FIG . 28 centeU . S . Patent -Aug . 15, 2017 -Sheet 18 of 24 US 9 ,731 ,012 B2 LPS +TGF-b1 LASER+LPS CONTROLLPS3J/cm230J CONJUGATEDBSA-TGFb1NPS LPS FIG.29B FIG.30B ND CONTROLCONTROL ND 450 40043507 300 250 200 1507-4100 50 0+ 900 800 700 600500 400 300 200 100- )104 * RFU- mL / pg ( 1b- IL ) 104 * RFU- mL / pg ( 1b -IL LPS +TGF-b1 LASER+LPS CONTROLLPS3J/cm230J CONJUGATEDBSA-TGFb1NPS LPSLPS FIG.29A FIG.30A CONTROL 800, 700-4 600 500 300 200 100 3000 2500 2000 1500 000 66)104 * RFU- mL / pg ( a- TNF ) 104 * RFU -mL / pg ( a -TNF U . S . Patent Aug . 15, 2017 Sheet 19 of 24 US 9 ,731 , 012 B2 100mg/ml 10mg/ml 2000FIG.31 atent Aug . 15 , 2017 Sheet 20 of 24 US 9 ,731 , 012 B2 NPPBS HOURS24 HOURS5 MIN90 MIN60 MIN45 MIN30 MIN25 MIN20 ??????? MIN15 MIN405 WNIWO BASELINE FIG.32A 2.50E+08 2.00E+08 0850E+.1 1.00E+08 5.00E+07 0.00E+00 )sr /cm2 /s /p ( RADIANCE AVG CONTROL:LATEXBEADS CARRAGEENAN 09710e+1.ROI2-09621e+ROI-5.1HOURS4 U . S . Patent Aug . 15, 2017 Sheet 21 of 24 US 9 ,731 ,012 B2 NP aPBS 7DAY 5DAY 3DAY 1DAY 0.6DAY FIG.32B ) sr/ cm2 /s / p( RADIANCE AVG CARRAGEENAN CONTROL:SELECTIN-BASED 4HOURS U . S . Patent Aug . 15, 2017 Sheet 22 of 24 US 9 ,731 ,012 B2 PBS-NP 7DAY 5DAY 3DAY 1DAY 0.6DAY 07 FIG.32C )sr / cm2 / s / p ( RADIANCE AVG NOTEXIN CONTROL:MPO-BASED 3DAYS atent Aug . 15 , 2017 Sheet 23 of 24 US 9 ,731 , 012 B2 DAYO DAY2 + 4HRS + 15MIN DAY3 SEED IMPLANT & INJECT LASER LUCIFERASE HIIIISCAFFOLD CARREGENNAN B1: BSANP IMAGING FIG . 33A FIG . 33B e TGFR1 BSA NP FIG . 33C Us Patentatent soAug . 15 , 2017we Sheetmus 24 of 24 US 9 ,731 , 012 B2 DAYO DAY5 DAY10 DAY12 IMPLANT INJECT B1- BSA NP OR PBS INJECT B1- BSA NP OR PBS SACRIFICE & PEI SCAFFOLD & LASER TREATMENT & LASER TREATMENT ANALYZE - FIG .