Lymphatic Route in Cardiovascular Medicine

Lymphatic Route in Cardiovascular Medicine: Comparison

Please note this is a comparison between Version 1 by Nolwenn Tessier and Version 2 by Peter Tang.

The lymphatic network is a unidirectional and low-pressure vascular system that is responsible for the absorption of interstitial fluids, molecules, and cells from the peripheral tissue, including the skin and the intestines. Targeting the lymphatic route for drug delivery employing traditional or new technologies and drug formulations is exponentially gaining attention in the quest to avoid the hepatic first-pass effect.

lymphatics
cardiovascular diseases
drug delivery route
nanotechnology

1. Introduction

Cardiovascular diseases (CVD) are one of the leading causes of death worldwide [1]. CVD include coronary heart disease, myocardial infarction (MI), heart failure (HF), stroke, and artery diseases [2]. Treatments for cardiovascular diseases are numerous, and the routes of administration are diverse. The chosen drug delivery route is a key determinant of the pharmacodynamics, pharmacokinetics, as well as toxicity of the delivered compounds. Yet, side effects or therapeutic failures are raising concerns, highlighting the need for new administration routes and improved formulation of molecules that reduce their degradation by hepatic metabolism. Drug delivery refers to the methods, approaches, or strategies employed for the transport of pharmaceutical compounds to an organism to achieve a desired therapeutic outcome. With this intent, various routes of administration are used to manage CVD and their risk factors, including parenteral (intravenous (IV), intradermal (ID), intramuscular (IM), subcutaneous (SC), and intraperitoneal (IP)), and transmucosal (oral, nasal, pulmonary, ocular, and genital) and transdermal route [3]. Drug absorption and transport through the lymphatic system makes it possible to avoid hepatic metabolism and is a privileged target in pathologies, such as particular types of cancer (chemotherapeutics [4]) or vaccines ^[5][6][5,6] (HIV [7]), but also for macromolecules [8], and the extensively hepatic-metabolized compounds ^[9][10][9,10].

2. Conventional and Novel Therapies to Treat CVD

Historically, small molecules have been used for the treatment of CVD. However, these molecules improve the symptoms and slow down the disease progression without having an actual regenerative effect on the affected tissues or organs ^[11][29]. Thus, the remaining unmet clinical needs necessitated the urgent seek for other potential therapeutic options.

Gene therapy is one of the most promising treatment strategies for CVD ^{[12][13][14][15][16]}[30,31,32,33,34], inherited or acquired, through targeting the causative genes engaged in the induction and progression of the disease. It works through replacing defective genes, silencing overexpressed ones or providing functional copies of specific therapeutic genes, thanks to DNA, RNA (siRNA, microRNA, mRNA), and antisense oligonucleotides (ASO) ^[17][35]. Back in the 1950s and 1960s, several attempts were made to directly transfect cells with DNA and RNA. Nevertheless, in vivo studies failed to show a noticeable success. Thus, selecting a suitable vector to deliver gene therapy is as important as selecting the agent itself ^[18][19][36,37]. Generally, vectors can be divided into viral and non-viral. The most commonly used viral vectors are retrovirus (RV), adenovirus (AV), adeno-associated virus (AAV), and lentivirus ^[20][38]. The most commonly used non-viral vectors include lipid-based vectors using cationic lipids and polymer-based vectors using cationic polymers ^[21][39]. Cationic lipids complex with the genetic materials to form lipoplexes or lipid nanoparticles (LNP), while cationic polymers form polyplexes ^[22][40]. In 2012, cardiovascular gene therapy was the third most common application for gene therapy (8.4% of the total gene therapy trials). However, clinically, it is still in the infancy stage, and a lot of effort is yet to be expended to correct the underlying basal molecular mechanisms behind different cardiovascular disorders ^[23][24][41,42].

3. Treating CVD through Various Administration Routes

3.1. Oral Administration

Among the various routes of administration, the oral route is the most commonly employed. It exhibits many advantages, including pain avoidance, ease of administration, patient compliance, reduced care cost, and low incidence of cross-infection. Furthermore, it is amenable to various types and forms of pharmaceuticals ^[25][48] (Table 1). While some drugs are intended to target the gastrointestinal tract (GIT), the majority are employed to exert a systemic therapeutic effect. Nevertheless, the oral bioavailability of most pharmaceutical compounds depends mainly on their solubility, permeability, and stability in the GIT environment ^[26][27][28][49,50,51].

Table 1. Oral delivery of various treatments for CVD.

Condition		Intervention and Identifier			Target				Intervention and Identifier		Target
Dose and Outcome
Diabetes		Metformin
Diabetes		Proinsulin peptide vaccine C19-A3			CD4 T cells		From 500 to 850 mg, 2–3 times a day, during the meal ^[29]	[58]
Three equal doses—10–100 µg	Vaccine was well tolerated ^[111		Diabetes			Sulfonylureas		Meglitinide			Dosage is very different from one class of medication to another ^[30]	[59]
Diabetes		Acarbose,		Miglitol		Voglibose			Carbohydrate digesting enzymes in the brush border		50 mg three times daily (up to 100 mg) ^[31]	[60]
Diabetes		Rosiglitazone		Pioglitazone			PPAR-α			Rosiglitazone: 4 mg per day (up to 8 mg)	Pioglitazone: 15–30 mg per day ^[32]	[ 61]

]. To target the lymphatic system exclusively, this type of injection must be combined with the use of macromolecules. As described in Table 2, subcutaneous injections are used as treatment for various conditions ^{[76][77][78][79][80][81][82][83][84][85][86][87][88][89][90][91][92][93][94][95][96][97][98][99][100][101][102][103]}[116,117,118,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143].

Table 2. Therapies targeting CVD using subcutaneous injection.

	Condition			Intervention and Identifier			Therapy			Target			Stage and Status			Dose and Outcome
	Diabetes			Insulin						Different types of insulin		At least 3 injections per day		Dosage adapted to the patient ^[ ^76]	[ 116 ]
^]		[	177 ]		Diabetes			Exenatide		Lixisenatide		Liraglutide		Exenatide LAR		Albiglutide		Dulaglutide						GLP-1 analogues ^[
	Diabetes			C19-A3		(NCT02837094)		⁷⁷		CD4 T cells		^]		Three doses—10 ug		In vitro and ex vivo studies of in human skin reported rapid diffusion of the injected particles through the skin layers and preferential uptake by Langerhans cells in the epidermis, which have a primary role in the tolerance mechanism ^[112]	[ 178]		[117]	Exenatide: 5–10 µg twice a day		Lixisenatide: 10–20 µg once daily		Liraglutide: 0.6–1.8 mg once daily		Exenatide LAR: 2 mg once a week		Albiglutide: 30–50 mg once a week		Dulaglutide: 0.75–1.5 mg once a week
	Diabetes
	Diabetes		Vaccine formed of virus-like particles coupled to IAPP				PIpepTolDC vaccine (NCT04590872)			Tolerogenic DC Vaccine		Against the insoluble IAPP- derived amyloid aggregates			One dose and another after 28 days		No results yet, but, it is believed to be able to produce proinsulin-specific Treg ^[113]	[	179 ]	Three doses—10 µg		Strong immune response against these aggregates and restored insulin production Diminished the amyloid deposits in the pancreatic islets, reduced the level of the pro-inflammatory cytokine IL-1β, and reprieved the onset of amyloid-induced hyperglycemia ^[78]	[ 118]
		Diabetes			IL-1β epitope peptide				Against IL-1β				Three doses—50 µg		Enhancement glucose tolerance, improved insulin sensitivity, restored β-cell mass, reduced β-cell apoptosis, and enhanced β-cell proliferation, as well as downregulation of IL-1β expression and inhibition of the inflammatory activity ^[79]^[80]	[ 119,120]		Diabetes			Sitaglipin		Vildaglipin		Saxaglipin		Linaglipin		Aloglipin
	Diabetes			hIL1bQb		vaccine		(NCT00924105)DPP4			2.5–100 mg once daily depending on the inhibitor used ^[33]	[62]
		Against IL-1β				Six doses—300 µg		Mediated a dose-dependent IL-1β-specific antibody response		More studies are required to precisely investigate the clinical efficiency of this vaccine ^[ ^81]	[ 121 ]		Diabetes
	Diabetes		Dapagliflozin		Canagliflozin		Empagliflozin			Neutralizing		antibodies against DPP4		SGLTP2			Dapagliflozin: 2.5–10 mg daily		Canagliflozin: 100–300 mg		Empagliflozin: 5–25 mg daily ^[		³⁴ ^]	[ 63 ]
	The GLP-1 and GIP inhibitor, DPP4				Five doses—2–20 µg		Increased pancreatic and plasma insulin level and improved postprandial blood glucose level ^[82]	[ 122]		Diabetes

DC: Dendritic cells; Treg: immunoregulatory T cells.

3.4. Intramuscular Injection

Intramuscular injections are used to target the deeper muscle tissue that is highly irrigated. This route of injection allows a rapid absorption and prolonged action. The medication would enter the bloodstream directly and, thus, allow the “bypass” of the hepatic metabolism. It is mainly used for the administration of vaccines ^[114][180] (hepatitis, flu virus, tetanus) or with specific pathologies, such as rheumatoid arthritis and multiple sclerosis. It is frequently performed in the upper arm ^[115][181] but also in the hip or thigh ^[116][182]. It is possible to administer up to 5 mL via this route, based on the site of injection ^[117][183]. As lymphatic vessels are present in the skeletal muscle and the connective tissue ^[118][184], this leads to the assumption the lymphatic system might be involved in the drug absorption following intramuscular administration. As presented in Table 4, several conditions are treated with this type of injection ^{[119][120][121][122]}[185,186,187,188].

Table 4. CVD therapies using intramuscular administration.

AAV1

SERCA2a

Phase IIb

(completed)

Single infusion of 1 × 10¹³ DRP of AAV1/SERCA2a

Phase IIb (CUPID-2b): no improvement was observed at the tested dose in patients with HF during the follow-up period

^[125]

[

201]

MYDICAR

(NCT01966887)

AAVI

SERCA2a

Phase II

(Terminated)

1 × 10¹³ DRP of AAV1/SERCA2a as a single intracoronary infusion

Phase II: no improvement observed in the ventricular remodeling.The study terminated driven by the CUPID-2 trial neutral outcome

^[128]

[

211]

SRD-001

(NCT04703842)

AAVI

SERCA2a

Phase I/II

(Active, not recruiting)

Single administration of 3 × 10¹³ vg

AG019

(NCT03751007) or in combination with the anti-CD3 monoclonal antibody teplizumab

2 or 6 capsules per day for 8 weeks (repeated dose) or for one day (single dose)

CUPID-3: aims to investigate the safety and efficacy of SRD-001 in anti-AAV1 neutralizing antibody-negative subjects with HFrEF

HTN

CVDhR32 vaccine

INXN-4001

(NCT03409627)

Non-viral, triple effector plasmid

Renin-derived peptide

SDF-1α,

S100A1,

VEGF-165

Phase I

(Completed)

Five doses—500 µg

Reduced systolic blood pressure by 15 mmHg

^[83]

[

123]

Single 80 mg dose, given in 40 mL or 80 mL at a rate of 20 mL/min

Phase I: an improvement in the quality of life in 50% of patients was reported

^[129]

[

212]

Diabetes

Insulin nanocarriers

Protection of insulin from enzymatic degradation

HTN

ACRX-100

(NCT01082094)

Enhancement of stability, intestinal permeability, and bioavailability

^[17]

Angiotensin I

vaccine (PMD3117)

Plasmid DNA

[35]

SDF-1

Phase I

(Completed)

Three or four doses—100 µg

The vaccine failed to reduce the blood pressure

^[84]

[

124]

Single escalating doses, injected at multiple sites

Preclinical studies: enhanced vasculogenesis and improved cardiac function reported with all doses

^[130]

[

213]

Diabetes

Electrostatically-complexed insulin with partially uncapped cationic liposomes

Improved insulin pharmacokinetic profile ^[35]

HTN

AngI-R vaccine

[64]

Modifiedendogenous angiotensin I peptide

JVS-100

(NCT01643590)

Plasmid DNA

SDF-1

Four doses—50 µg

15 mmHg reduction in systolic blood pressure and reduced angiotensin I/II level

Phase II

(Completed)

^85]

[

125]

Single injection of escalating doses (15 and 30 mg)

Phase II (STOP-HF): JVS-100 showed potential to improve cardiac function through reducing left ventricular remodeling and improving ejection fraction

^[131]

[

214]

Diabetes

Insulin-loaded PLGA

Improved bioavailability and sustained hypoglycemic effect

HTN

ATRQβ-001

JVS-100

(NCT01961726)

Plasmid DNA

^[36]

[65]

Angiotensin II type I receptors

SDF-1

Phase I/II

(Unknown)

Two doses—100 µg

Protective role against target organ damage induced by hypertension

^[86]

[

126]

Single injection of escalating doses (30 and 45 mg)

Phase I (RETRO-HF): JVS-100 showed promising signs of clinical efficacy

^[132]

[

215]

Diabetes

Exenatide combined to phase-changeable nanoemulsion with medium-chain fatty acid

HTN

Enhancement of intestinal absorption and lymphatic transport ^[

ATR12181 vaccine

AZD8601

(NCT02935712)^37]

(NCT03370887)

mRNA

[66]

Angiotensin II type I receptors

VEGF-A165

Phase IIa

(Active, not recruiting)

Nine doses—0.1 mg

Attenuated the development of hemodynamic alterations of hypertension

^[87]

[

127]

Single injection of escalating doses (3 mg and 30 mg)

Preclinical studies: promoted angiogenesis, improved cardiac function and enhanced survival were reported

^[133]

[

216]

Phase I: ID injection of AZD8601 was well tolerated and enhanced the basal skin blood flow

^134]

[

217

HTN

Prazosine Terazosine Doxazosine

Alpha-adrenergic receptor

Prazosine: 3–7.5 mg per day in two doses

]

HTN

Terazosine: 1–9 mg per day in the evening at bedtime

Doxazosine: 4 mg per day

^38]

[

]

CYT006-AngQb vaccine

Against angiotensin II

NAN-101

(NCT04179643)

100 or 300 µg

Reduction in blood pressure and reduced ambulatory daytime blood pressure

^[88]

AAV

I-1c

Phase I

(Recruiting)

[128]

Single escalating doses (3 × 10¹³ vg–3 × 10¹⁴ vg) of NAN-101

Preclinical studies: enhancement in left ventricular ejection fraction and improved cardiac performance

^[135]

[

218]

HTN

Clonidine Methyldopa

Alpha-adrenergic receptor (agonists)

Clonidine: 0.1 mg twice daily

HTN

Ang II-KLH

vaccine

AMI IHD

VM202RY

(NCT01422772)

(NCT03404024)

DNA plasmid

^39]

[72]

Methydopa: 250 mg two to three times daily

^[40]

[

HGF-X7

Phase II

(Recruiting)

Single escalating (0.5–3 mg) doses, administered into multiple sites

Phase I: improved myocardial function and wall thickness

^[136]^[137]

[

219,220]

Angina pectoris

AdVEGF-D (NCT01002430)

VEGF-D

Phase I/IIa

(Completed)

Single escalating (1 × 10⁹–1 × 10¹¹ Vpu) doses, injected into multiple sites in the endocardium

Phase 1/IIa: AdVEGF-D improved myocardial perfusion reserve in the injected region

^[137]

[

220]

Ad-HGF

(NCT02844283)

Condition

			Target			Dose and Outcome
Diabetes		Preproinsulin-encoding plasmid DNA			Pancreatic islets			40% higher survival rate as compared to the control group ^[119]	[185]
HTN		CoVaccine HT		(NCT00702221)			Against angiotensin II			Three doses		Terminated in 2016 due to dose-limiting adverse effects
HTN		AGMG0201		vaccine			Against angiotensin II			High or low dose (0.2 mg plasmid DNA and 0.5 or 0.25 mg Ang II-KLH conjugate) Ongoing
ACS	HF		CVD			Inactivated influenza vaccine				Less frequent hospitalization from ACS, hospitalization from HF and stroke ^[120]	[186]
MI		Influenza vaccine				Risk of cardiovascular-related death was significantly lower ^[121]	[187]
CVD	MI			Pneumococcal vaccines				Reduced incidence of cardiovascular events and mortality		Reduced risk of MI in the elderly ^[122]	[ 188]
MI	HF		Stroke			Influenza vaccine		(NCT02831608)				The primary endpoints: death, new MI and stent thrombosis	Secondary endpoints: patients with hospitalization for HF

HTN: Hypertension; AngII-KLH: Angiotensin II—keyhole-limpet hemocyanin; ACS: Acute coronary syndrome; CVD: cardiovascular disease; HF: Heart failure; MI: Myocardial infarction.

3.5. Intramyocardial Injection

Direct intramyocardial injection is the most effective and commonly used way for gene delivery to the heart owing to its ability to achieve a high concentration of the injected compound at the injection site ^[123][207]. It is a preferential route to directly target lymphatic vessels due to their high density in the myocardium ^[104][124][159,208]. Various CVD and their treatments via intramyocardial injection are presented in Table 5 ^{[125][126][127][128][129][130][131][132][133][134][135][136][137][138][139][140][141]}[201,209,210,211,212,213,214,215,216,217,218,219,220,221,222,223,224].

Table 5. Use of intramyocardial injections in several therapies targeting CVD.

	Condition			Intervention and Identifier			Therapy			Target			Stage and Status			Dose and Outcome
	HF			Ad5.hAC6		(NCT007)			Ad5			AC6			Phase I/II		(Completed)			Single administration of escalating doses (3.2 × 10⁹ vp to 10¹² vp)		Phase II: Reduced HF admission rate. Enhanced left ventricular function beyond the optimal HF therapy following a single administration ^[126]	[ 209]
	HF			Ad5.hAC6		(NCT03360448)			Ad5			AC6			Phase III		(withdrawn)			Phase III: withdrawn for re-evaluation
	HF			MYDICAR		(NCT00454818)			AAV1			SERCA2a			Phase I/II (Completed)			Single administration of escalating doses (1.4 × 10¹¹–1 × 10¹³ DRP of AAV1/SERCA2a)		Phase I/II (CUPID): high-dose treatment resulted in increased time and reduced frequency of cardiovascular events within a year and reduced cardiovascular hospitalizations ^[127]	[ 210]
	HF			MYDICAR		(NCT01643330)

73
]


Angiotensin II
			Three doses—5 µg		Suppressed post-MI cardiac remodeling and improved cardiac function ^[89]	[ 129]
	HTN			Carvedilol into nanoemulsion			Beta-adrenergic receptors
	MI			Celecoxib loaded in nanoparticles			Significant improvement in its absorption, permeability, and bioavailability ^[41]^[42]	[88,89]
			Promoted vascularization in the ischemic myocardium and delayed HF progression	^[	^90]	[130]		HTN			Valsartan, Ramipril and Amlodipine into nanoemulsion
	MI			Chitosan-hyaluronic acid based hydrogel containing deferoxamine-PLGA		nanoparticles		Enhanced solubility, oral bioavailability, and pharmacological outcome ^[43		^]	[90]
			HGF			Phase I/II (Unknown)		Persistent neovascularization in mice ^[91]	[131]		Single dose		Preclinical studies: Ad-HGF preserved cardiac function, reduced infarct size, and improved post-MI cardiac remodeling ^[138][221]; fractional repeated dosing significantly improved cardiac function compared with single injection ^[139]	[ 222]		HTN			Felodipine-loaded PLGA nanoparticles			Calcium-channel			Sustained drug release both in vitro and ex vivo ^[44]	[93]
	HCL
	MI		Alirocumab		Evolocumab				PCSK9			L-type Ca²⁺ channels’ AID peptide and antioxidant molecule (curcumin) in poly nanoparticles					One dose every two weeks ^[92]^[93]	[132,133]		Reduced the elevated level of ROS and the intracellular Ca²⁺ ^[140]	[223]		MI		HF		HTN		Arrhythmia	ß-blocker	Beta-adrenergic receptors	Acebutol: 200 mg twice daily
	HCL
	LPLD		Inclisiran			Alipogene tiparvovec		(NCT00891306)		^[45]			AAV			[74]
	PCSK9				LPL			Approved		Two doses per year ^[94]	[134]		1 × 10¹² GC/kg		Phase II/III: reduction in mean total plasma and chylomicron TG level ^[141]	[ 224]		MI		HF		HTN
	HoFH		HeFH		severe HCL		Conversion enzyme		inhibitors			Mipomersen		(NCT00607373)		(NCT00706849)		(NCT00770146)		(NCT00794664)		Conversion enzyme				ASO		Captopril: 100 mg per day ^[46]	[75]
	ApoB			Approved			200 mg once/week.		Phase III: reduction in LDL-C ^[95]	[ 135]		MI		HF		HTN			Valsartan		Losartan			Angiotensin II
	ASCVD HCL HeFH			Inclisiran		(NCT03399370)		(NCT03400800)		(NCT03397121)		20 mg twice a day, up to 160 mg ^[47]	[76]
	siRNA			PCSK9			Approved			284 mg inclisiran, injected on day 1, day 90 and then twice/year		Phase III: reduction in LDL-C level ^[94]^[96]	[ 134,136]		HF		HTN			Hydrochlorothiazide		Bumetanide			Angiotensin/neprilysin receptor
	FCS			Volanesorsen		(NCT02211209)		49 mg/51 mg twice daily and doubled after 2–4 weeks ^[48]	[77]
	ASO			ApoC3			Approved			300 mg once/week		Phase III: reduction in mean plasma APOC3 and TG level ^[97]	[ 137]		HF		HTN			Sacubitril		Valsartan			Calcium channel
	Elevated LP(a)			ISIS-APO(a)Rx		(NCT02160899)		5–10 mg daily		ASO		^[49]	[78]	60 mg three times daily ^[50]	[ 79]
	APO(a)			Phase II (Complete)			Multiple escalating (100–300 mg) doses, injected on a weekly interval for 4 weeks each		Phase I/II: reduction in plasma Lp(a) concentration ^[98]	[ 138]		HTN		Arrhythmia			Amlodipine		Diltiazem			Calcium channel			5–10 mg daily ^[
	Elevated LP(a)		CVD			AKCEA-APO(a)-LRx		(NCT03070782)		(NCT02414594)		(NCT04023552)		^49]		GalNAc3		conjugated-ASO[78]		60 mg three times daily ^[50]	[ 79]
		APO(a)			Phase III		(Recruiting)			80 mg administered monthly		Phase I/II: reduction in plasma Lp(a) ^[98]	[ 138]		HF
	HTG		CVD		FCS		Ivabradine				AKCEA-APOCIII-LRx		(NCT02900027)		(NCT03385239)		(NCT04568434)		Bradycardic		5–7.5 mg twice a day ^[		GalNAc3		conjugated-ASO		⁵¹ ^]	[ 80]
	APOC3			Phase III		(Recruiting)			Multiple dosing injected as once/4 weeks for up to 49 weeks		Phase II: reduction in ApoC3 and TG levels ^[99]	[ 139]		HF		MI
	HTG		FH		HLP		Eplerenone		Spironolactone				Vupanorsen		(NCT02709850)		(NCT04459767)		(NCT04516291)		Aldosterone			ASO		50 mg once a day ^[52][81] and 12.5–25 mg with each intake ^[53]	[82]
	ANGPTL3			Phase IIb		(Active, Not recruiting)			Multiple escalating dosing (60–160 mg, once/2 or 4 weeks)		Phase I: reduction in TG and LDL-C levels ^[100]	[ 140]		HF		Arrhythmia			Digoxin				0.25 mg once daily ^[54]
	HCL			Neutralizing antibodies against PCSK9			[83	]
	PCSK9				Three doses—5–50 µg		Long-lasting reduction in the level of total cholesterol, VLDL and		chylomicron ^[ ^101]	[ 141 ]		HF		MI		HCL			Statin			HMG-CoA
	HCL			10 mg once daily ^[		AT04A		^55]	[84]
		PCSK9				Five doses		Strong and persistent anti-PCSK9 antibody production, reduced plasma cholesterol level, attenuated progression of atherosclerosis and reduced vascular and systemic inflammation ^[102]	[ 142]		MI			Aspirin			Platelets			325 mg, then 81 mg per day ^[56]	[
	HCL			AT04A	85]
		PCSK9				Four doses—15 µg and 75 µg		Reduced serum LDL-C level and elevated anti-PCSK9 antibody titer ^[103]	[ 143]		MI
	HCL		Clopidogrel		Prasugrel		Ticagrelor				A peptide representing the mouse ANGPTL3		Platelets			300 mg, then 75 mg daily with aspirin		60 mg, then 10 mg daily		180 mg, then 90 mg twice a day ^[ ^57]^[58]			Angiopoietin-like proteins 3 (ANGPTL3)	[ 86 ,87]
			Three doses—5 µg			HCL			Ezetimibe			Intestinal cholesterol absorption			10 mg once daily ^[59]	[99]
Reduced steady-state plasma TGs and promoted LPL activity			HLD			Tricor		Triglide				Fenofibrates 100–300 mg per day ^[60]	[100]
	HCL		HLD			Atorvastatin formulated into ethylcellulose nanoparticles				Enhanced atorvastatin’s lymphatic absorption and oral bioavailability ^[61]	[101]
	HCL		HLD			Atorvastatin formulated into nanocrystals prepared with poloxamer 188				Improved atorvastatin’s gastric solubility and bioavailability ^[62]	[102]	Reduced circulating cholesterol, TG and LDL
	HCL		HLD			Atorvastatin formulated into polycaprolactone nanoparticles				Enhanced atorvastatin’s bioavailability ^[63]	[103]
	HCL		HLD			Nanostructured lipid carriers				Enhanced atorvastatin bioavailability by 2.1 fold compared to the commercial product: lipitor^®		Reduced the serum level of cholesterol, TG and LDL ^[64]	[ 104]
	HCL		HLD			Nanoemulsion				Increased the bioavailability of atorvastatin compared to the commercial tablet ozovas^TM ^[65]	[105]
	HCL		HLD			Simvastatin		Rosuvastatin		Fluvastatin		Fibrates		Ezetimibe		lipid-based		nanoparticles				Improved bioavailability via lymphatic uptake ^[66]^[67]^[68]^[⁷³^]^[⁷⁴^]	[106,107,108^69],109^[70],110^[71],111^[72],112^[,113,114]

PPAR- α: peroxisome proliferator-activated receptor- α; DPP4: dipeptidyl peptidase-4; SGLTP2: Sodium glucose co-transporter-2; PLGA: Poly lactic-co-glycolic acid; HTN: Hypertension; MI: Myocardial infarction; HF: Heart failure; HCL: Hypercholesterolemia; HMG-CoA reductase: Hydroxymethyl glutaryl coenzyme A reductase; HLD: Hyperlipidemia; TG: Triglycerides; LDL: Low density lipoprotein.

3.2. Subcutaneous Injection

Subcutaneous injections consist of injecting a molecule under the dermis, in the SC cell layer (interstitial space), and slightly before the muscle, mostly in the abdomen or thigh. The injected molecules will, therefore, either be degraded or phagocytized at the site of injection and join the lymphatic system or the bloodstream ^[75][115

GLP-1: glucagon-like peptide-1; IAPP: Islet amyloid polypeptide; DPP4: dipeptidyl peptidase-4; GIP: glucose-dependent insulinotropic polypeptide; HTN: Hypertension; HF: Heart failure; MI: Myocardial infarction; HCL: Hypercholesterolemia; HoFH: Homozygous familial hypercholesterolemia; HeFH: Heterozygous familial hypercholesterolemia; AngII-KLH: Angiotensin II—keyhole-limpet hemocyanin; PCSK9: Proprotein convertase subtilisin/kexin type 9; ASO: Antisense oligonucleotides; ApoB: Apolipoprotein B; LDL-C: low density lipoprotein cholesterol; ASCVD: Atherosclerotic cardiovascular disease; FCS: Familial chylomicronemia syndrome; TG: Triglycerides; LP(a): Lipoprotein(a); APO(a): Apolipoprotein (a); CVD: Cardiovascular diseases; GalNAc3: Triantennary N-acetyl galactosamine; HTG: Hypertriglyceridemia; FH: Familial hypercholesterolemia; HLP: Hyperlipoproteinemia; ANGPTL3: Angiopoietin-like proteins 3; VLDL: Very low density lipoprotein; LPL: Lipoprotein lipase.

3.3. Intradermal Injection

Lymphatic capillaries are present in the dermis and, thus, preferentially take up the injected molecules. Unlike the blood capillaries, initial lymphatics lack the basement membrane underlying the endothelial layer. The distal part of initial LV is exclusively composed of LECs with button-like junctions ^[104][159], leading to capillaries that have inter-endothelial gaps with size ranges from a few nanometers to several microns ^[4][105][4,160]. Small particles (<10 nm) [4] and medium-sized macromolecules (up to 16 kDa) ^[106][161] are mainly transported away from the interstitial spaces by blood capillaries, thanks to mass transport ^[107][108][162,163]. In contrast, lymphatic access of large particles with diameters exceeding 100 nm is hindered by their restricted movement through the interstitium, via diffusion and convection [4]. In between, particles with a size of 10–100 nm [4] and macromolecules with a size of 20–30 kDa ^[106][161] show preferential uptake into the highly permeable lymphatic capillaries either passively (paracellular) or actively (transcellular) through the lymphatic endothelial cells ^[109][164]. Indeed, it has been shown that the optimal diameter to target the lymphatic vessels in the dermis is 5 to 50 nm in mice ^[110][165].

Table 3 presents several vaccines used for diabetes through intradermal injection ^{[111][112][113]}[177,178,179].

Table 3. Intradermal administration as treatment for diabetes.

	Condition

HF: Heart failure; hAC6: Human adenylyl cyclase type 6; vp: Virus particles; AAV: Adeno-associated virus; SERCA2a: Sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase; DRP: DNase-resistant particles; HFrEF: HF with reduced ejection fraction; CVD: Cardiovascular diseases; SDF-1a: stromal cell-derived factor 1; VEGF: Vascular endothelial growth factor; I-1c: Constitutively active inhibitor-1; vg: Viral genomes; AMI: Acute myocardial infarction; IDH: Ischemic heart disease; HGF-X7: Hepatocyte growth factor-X7; AV: Adenovirus; Vpu: Viral protein U; HGF: Hepatocyte growth factor; AID: alpha-interacting domain; ROS: reactive oxygen species; LPL: Lipoprotein lipase; TG: Triglycerides; GC: Genome copies.

3.6. Intravenous Injection

Intravenous injections are often used for rehydration, nutrition, and therapeutic treatments (for example, blood transfusion or chemotherapy), as well as to avoid hepatic metabolism ^[142][230]. The interest of this route of administration is the continuous treatment, or regular frequencies, by the installation of a catheter ^[143][231]. However, the lymphatic system is only scarcely involved following IV injections ^{[144][145][146]}[232,233,234]. Table 6 presents several conditions treated with this type of injection ^{[45][54][56][147][148][149][150][151][152][153][154][155][156][157][158][159][160]}[74,83,85,235,236,237,238,239,240,241,242,243,244,245,246,247,248].

Table 6. Intravenous administration of medication as treatment for CVD.

Condition

	Therapy			Target			Stage and Status			Dose and Outcome
HTN		NO-releasing nanoparticles						Reduction in the mean arterial blood pressure ^[147]	[235]
HF	Arrhythmia			Digoxin						Dose: 0.25 mg once daily ^[54]	[83]
MI	HF		HTN		Arrhythmia			ß-blocker				Beta-adrenergic receptors				Acebutol: 200 mg twice daily ^[45]	[74]
HF		Mesoporous silicon vector (Nanoconstruct)						Able to internalize, accumulate, and traffic within the cardiomyocytes ^[148]	[236]
HF		Combination of biocompatible magnetic nanoparticles and low-frequency magnetic stimulation				Cardio-myocytes				Managed the drug release by controlling the applied frequencies ^[149]	[237]
HF		S100A1-loaded nanoparticles, decorated with N-acetylglucosamine						Regulated Ca²⁺ release and restored contractile function in the isolated failing cardiomyocytes ^[150]	[238]
HF		Biodegradable nanoparticles conjugated with myocyte-targeting peptide and PDT-enabling photosensitizer			PDT			Cardio-myocytes				Induced cell-specific death upon application of laser light, leaving adjacent and surrounding cells completely intact ^[151]	[239]
MI		Unfractionated		heparin						Anticoagulant		60 IU/kg for initial bolus		12 IU/kg/h for maintenance ^[ ^152]	[ 240 ]
MI		Aspirin				Platelets				325 mg, then 81 mg per day ^[56]	[85]
MI		Human recombinant VEGF-165						Significant improvement in the infarcted zone perfusion and cardiac function for up to six weeks post-MI ^[153][241].
MI		Nanoparticles containing siRNA						Anti-inflammatory effect in the infarcted heart and reduction of the post-MI heart failure ^[154]	[242]
MI		Magnetic nanoparticles-loaded cells						Robust improvement in the left ventricular and cardiac function ^[155]	[243]
MI		Insulin-like growth factor electrostatically-complexed with PLGA nanoparticles						Higher incidence in preventing cardiomyocytes’ apoptosis, reducing infarct size, and enhancing left ventricular function ^[156]	[244]
MI		Pitavastatin in PLGA nanoparticles						Cardioprotective effect against ischemia-reperfusion injury ^[157]	[245]
HoFH		AAV8.TBG.HldlR		(NCT02651675)			AAV			hLDLR			Phase I/II (Completed)			Single dose		Preclinical studies: reduction in total cholesterol ^[158]^[159]	[ 246,247]
Elevated LDL-C		ALN-PCS02		(NCT01437059)			siRNA			PCSK9			Phase I		(Completed)			Single escalating (15 and 400 μg/kg) doses		Phase I: reduction in the level of circulating PCSK9 protein and LDL-C ^[160]	[ 248]

HTN: Hypertension; NO: nitric oxide; HF: Heart failure; MI: Myocardial infarction; PDT: Photodynamic therapy; VEGF: Vascular endothelial growth factor; PLGA: Poly lactic-co-glycolic acid; AAV: Adeno-associated virus; HoFH: Homozygous familial hypercholesterolemia; hLDLR: Human low density lipoprotein receptor; TBG: Thyroxine-binding globulin; LDL-C: low density lipoprotein cholesterol.

3.7. Intraperitoneal Injection

Intraperitoneal administration, in which therapeutic compounds are injected directly into the peritoneal cavity, is another attractive approach of the parenteral extravascular strategies. It is used specifically for the local treatment of peritoneal cavity disorders, e.g., peritoneal malignancies and dialysis. The peritoneal cavity contains the abdominal organs and the peritoneal fluid, normally composed of water, proteins, electrolytes, immune cells, and other interstitial fluid substances ^[161][272]. The high absorption rate associated to IP administration is promoted by the vast blood supply to the peritoneal cavity, along with its large surface area, which is further increased by the microvilli covering the mesothelial layer ^[162][273]. Injected compounds can enter the circulatory system after IP injection via both blood and lymphatic capillaries draining the peritoneal submesothelial layer ^{[162][163][164]}[273,274,275]. Besides, the peritoneal absorption of molecules is greatly affected by their physicochemical characteristics. This route of administration also allows for the injection of large volumes (up to 10 mL) ^[162][273]. Extensive experimental studies carried out on animals have revealed that the peritoneal cavity has favorable absorption of lipophilic and unionized compounds ^[165][276]. This type of injection is most exploited for preclinical studies, since it is the simplest to perform, especially in small animals and with little impact on the animals’ stress ^[162][166][273,277]. IP use in humans is limited, despite showing many benefits in previous studies and even being recommended, for certain types of chemotherapy, by the National Cancer Institute ^{[167][168][169]}[278,279,280].